Plasmodium Falciparum Histone Deacetylases As Novel Antimalarial Drug Targets

Plasmodium Falciparum Histone Deacetylases as Novel Antimalarial Drug Targets

Author Tran, Thanh Nguyen

Published 2010

Thesis Type Thesis (PhD Doctorate)

School School of Health Science

DOI https://doi.org/10.25904/1912/2472

Downloaded from http://hdl.handle.net/10072/367456

Griffith Research Online https://research-repository.griffith.edu.au Plasmodium falciparum Histone Deacetylases as Novel Antimalarial Drug Targets

Thanh Nguyen Tran 2009 Plasmodium falciparum Histone Deacetylases as Novel Antimalarial Drug Targets

Thanh Nguyen Tran Bachelor of Science, Honours I

Griffith Medical Research College Griffith Health Griffith University

Submitted in fulfilment of the requirements of the degree of Doctor of Philosophy

December 2009

Statement of Originality

This work has not previously been submitted for a degree or diploma in any university. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made in the thesis itself.

______

Thanh Nguyen Tran

December 2009

Acknowledgement

I would like to firstly thank Kathy and Tina for their patience and advice over the past four years.

Thanks to Don for his expert advice on P. falciparum transfection. Thanks to Dr. Deb Stenzel for her expert advice and help with Transmission Electron Microscopy imaging of P. falciparum parasites. I would like to acknowledge everyone that were involved in this project including Prof.

David Fairlie, Dr. Andrew Lucke, Dr. Bob Reid, Dr. Geoff Dow and Dr. Glen Boyle.

I also like to acknowledge funding sources from the ANZ PhD Trustee Medical Research

Scholarship, the QIMR PhD Top-up Student Scholarship, and the Australian Society for

Parasitology Travel Award Scholarship.

Many thanks go to everyone from the Malaria Biology Laboratory, the Scabies Mite Laboratory and the Clinical Tropical Medicine laboratory for being such a fantastic bunch of people to work alongside. To Simone, Linh and Anita – you are the most awesome friends. Finally, I would like to thank my mun and brother Ken for their unconditional supports.

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Publications by the Candidate Relevant to the Thesis

1. Andrews KT, Tran TN*, Lucke AJ, Kahnberg P, Le GT, Boyle GM, Gardiner DL, Skinner-

Adams TS, Fairlie DP: Potent antimalarial activity of histone deacetylase inhibitor analogues.

Antimicrobial Agents and Chemotherapy 2008, 52(4):1454-1461.

*Co-first Author: Equal contribution to manuscript

2. Dow GS, Chen Y, Andrews KT, Caridha D, Gerena L, Gettayacamin M, Johnson J, Li Q,

Melendez V, Obaldia N, 3rd, Tran TN, Kozikowski AP: Antimalarial activity of phenylthiazolyl- bearing hydroxamate-based histone deacetylase inhibitors. Antimicrobial Agents and Chemotherapy

2008, 52(10):3467-3477.

3. Andrews KT, Tran TN, Wheatley NC, Fairlie DP: Targeting histone deacetylase inhibitors for anti-malarial therapy. Current Topics in Medicinal Chemistry 2009, 9(3):292-308.

Manuscripts attached following list of references.

Abstract

Histone deacetylases (HDACs) are recognised as potential drug targets for many diseases including cancer, inflammatory diseases and some parasitic diseases including malaria. In eukaryotic cells, these enzymes play an important role in transcriptional regulation through modification of chromatin structure. Inhibitors of mammalian HDAC enzymes including trichostain A and apicidin are active against P. falciparum parasites, however these compounds are not selective for malaria parasites versus normal cell lines. The aims of this study were to examine the antimalarial potential of new hydroxamate-based HDAC inhibitors and to investigate a P. falciparum HDAC, PfHDAC1, as a potential new antimalarial drug target.

A panel of fourteen synthetic non-peptidic HDAC inhibitors derived from L-cysteine (L-CYS) and

2-aminosuberic acid (2-ASA) was investigated for antimalarial activity. All fourteen compounds display potent inhibitory activity against different P. falciparum strains (IC50 range 13 – 339nM) and some are up to 100 times more selective in killing parasites versus mammalian cells. In general, the 2-ASA derived compounds are more parasite selective than the L-CYS compounds (selective index (SI) 4 – 167 for 2-ASA compounds versus 2 – 13 for L-CYS compounds). One representative compound from the 2-ASA series (2-ASA-9) was examined for in vivo activity and was found to significantly suppress parasitemia in mice infected with P. berghei when administered orally at 10 mg/kg, twice a day, for three days (P <0.05). Likewise, pharmacokinetic studies with another 2-

ASA compound (2-ASA-14) confirmed that this compound is orally bioavailable (Cmax =

340ng/mL; Tmax = 1 h), but has a short half life (T1/2 ≤ 2 h).

To assess the potential of combining 2-ASA compounds with clinically used antimalarials, 2-ASA-

9 was tested in in vitro drug combination studies. 2-ASA-9 is antagonistic when combined with chloroquine (I = -4.44, P < 0.0001) or artemisinin (I = -0.92, P < 0.005), while combinations of

v quinine and 2-ASA-9 (I = -1.52, P = 0.1) resulted in a mild antagonistic interaction suggesting this combination may have potential in vivo antimalarial activity. Similar results were obtained with a control HDAC inhibitor, (SAHA; I = -1.31, P = 0.1).

To better understand when and how HDAC inhibitors act on malaria parasites, stage specific growth inhibition and mode of action assays were carried out. The HDAC inhibitors 2-ASA-9, 2-

ASA-14 and SAHA were all found to preferentially inhibit the in vitro growth of trophozoite compared to ring-stage parasites, and to act quickly on trophozoites (2 – 6 h). Like SAHA, the 2-

ASA compounds appear to be acting as HDAC inhibitors in the parasites. 2-ASA-9, 2-ASA-14 and

SAHA all cause hyperacetylation of parasite histone H4, which is a marker of HDAC inhibition in higher eukaryotic cells. Likewise, all three compounds inhibit deacetylase activity in P. falciparum nuclear extracts (IC50 100-155 nM). Another marker of HDAC inhibition in eukaryotic cells is alteration in transcription of up to 20% of genes. Preliminary studies confirm that HDAC inhibitors also affect the transcription of at least one P. falciparum gene, alpha-II-tubulin. Together these data support HDAC inhibition as a mode of action of these compounds in malaria parasites.

Class I/II HDAC enzymes play an important role in transcription regulation and hence represent an important target for cancer therapy and inflammatory diseases. To better understand PfHDAC1 as a potential antimalarial drug target, phylogenetic, in silico, and molecular studies were carried out.

Phylogenetically, PfHDAC1 is most similar to class I HDACs homologues from other apicomplxan parasites such as T. gondii (~73%) and Cryptosporidium (~65% amino acid identity), however this

P. falciapurm HDAC shares up to 50% identity with HDAC1 from mouse and human. A comparison of PfHDAC1 with HDAC8 from humans and the bacterial histone deacetylase-like protein, was carried out by generating an in silico homology model. The homology model of

PfHDAC1 showed that this protein has a conserved zinc binding active site similar to the bacterial histone deacetylase-like protein and human HDAC8. The surface lining the entrance to the active

vi site of PfHDAC1 contains three potential binding pockets and the entrance to the active site of

PfHDAC1, which has a relatively open binding site, indicates these features may be able to be exploited to develop parasite-selective compounds. Ligand docking studies demonstrated L-CYS and 2-ASA derivatives have binding affinity between 1µM – 1nM for PfHDAC1 which correlates well with experimental in vitro growth inhibition data.

To determine whether PfHDAC1 is essential to P. falciparum development, attempts to genetically disrupt Pfhdac1 were carried out. Disruption of Pfhdac1 was unsuccessful indicating this gene may be essential to the parasite and may represent a good parasite drug target. Furthermore, transgenic

P. falciparum parasites overexpressing PfHDAC1 fused to cmyc and green fluorescent protein

(GFP) were generated to confirm the subcellular localization of this protein in the parasite and as a tool to examine if overexpressed PfHDAC1 can alter the drug susceptibility profile to different

HDAC inhibitors. Expression of PfHDAC1 transgene was confirmed by Northern blot and Western blot. The transgenic parasites display similar drug sensitivity to different HDAC inhibitors as wildtype parasites and overexpression of PfHDAC1 does not change total deacetylase activity.

Interestingly, apparent down-regulation of PfHDAC1 occurs in overepression mutants indicating expression of this protein is tightly regulated in P. falciparum. Together, these findings indicate

PfHDAC1 may be essential to P. falciparum development.

Overall, this study has contributed to our understanding of the modes of action of HDAC inhibitors in P. falciparum parasites. This information will be useful in guiding the development of next generation of compounds with better parasite-selectivity and potency to current HDAC inhibitors.

HDAC inhibitors are exciting new antimalarial agents as they are acting on a different parasite target to existing antimalarials and may be useful new agents to address the ongoing issue of drug resistance.

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Table of Contents

Title Page i

Statement of Originality ii

Acknowledgement iii

Publication by the Candidature Relevant to the Thesis iv

Abstract v

Table of Contents viii

List of Figures xiii

List of Tables xvi

List of Abbreviations xvii

Chapter 1. Introduction 1

1.1 Malaria – Overview 2

1.2 Plasmodium lifecycle 3

1.3 Malaria parasite drug resistance 5

1.4 Approaches to drug discovery for malaria 6

1.5 P. falciparum histone deacetylases as potential new antimalarial drug targets 8

1.6 HDACs: function and characterization 9

1.7 Classification and function of eukaryotic HDACs 10

1.7.1 Key features of class I HDACs 10

1.7.2 Key features of class II HDACs 12

1.7.3 Key features of class III HDACs 13

1.8.1 P. falciparum class I and II HDACs 13

1.8.2 P. falciparum Sir2 HDACs 16

1.9 HDAC inhibitors as anticancer drugs 17

1.10 HDAC inhibitors as antimalarial agents 19

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1.11 Summary, hypotheses, and aims 23

Chapter 2. General Materials and Methods 24

2.1 P. falciparum in vitro cultures 25

2.2 In vitro P. falciparum growth inhibition assays 25

2.3 Blood and sera preparation 26

2.4 Cryopreservation of P. falciparum infected erythrocytes 26

2.5 Thawing of P. falciparum infected erythrocytes 26

2.6 Sorbitol synchronization 27

2.7 Saponin lysis 27

2.8 Nuclear protein extracts 27

2.9 Protein quantification 28

2.10 Deacetylase activity assays 28

2.11 Western blot 28

2.12 Stripping PVDF (Western blot) membranes 29

2.13 RNA extraction 29

2.14 Northern blot 30

2.15 Genomic DNA extraction 30

2.16 Polymerase chain reaction (PCR) 31

2.17 DNA sequencing 31

2.18 pGEM-T ligation 32

2.19 Plasmid extraction 32

2.20 Restriction digestion 32

2.21 DNA gel electrophoresis 33

2.22 Gel purification 33

2.23 E. coli transformation 33

2.24 P. falciparum transfection 34

2.25 Immunofluorescent assays (IFA) 34

Chapter 3. In vitro and in vivo antimalarial activity of L-cysteine and 2-aminosuberic acid HDAC inhibitor analogues 36

3.1 Introduction 37

3.2 Materials and methods 38

3.2.1 Compounds 38

3.2.2 P. falciparum in vitro growth inhibition assays 38

3.2.3 Mammalian cell toxicity assays 39

3.2.4 Oral pharmacokinetic studies in mice 39

3.2.5 In vivo antimalarial studies in mice 40

3.2.6 Isobologram assays 40

3.3 Results 41

3.3.1 In vitro antimalarial activity of new hydroxamate-based HDAC inhibitor

compounds based on L-CYS and 2-ASA structures 41

3.3.2 Oral bioavailability of 2-ASA-14 45

3.3.3 In vivo antimalarial efficacy of 2-ASA compounds in a mouse malaria model 45

3.3.4 In vitro drug interactions of HDAC inhibitors and known antimalarial drugs 46

3.4 Discussion 48

Chapter 4. Effects of hydroxamate-based HDAC inhibitors on P. falciparum

Parasites 53

4.1 Introduction 54

4.2 Materials and methods 54

4.2.1 Stage specific growth inhibition assays 54

4.2.2 Transmission electron microscopy (TEM) of HDAC inhibitor treated parasites 55

4.2.3 Histone hyperacetylation assays 56

4.3 Results 57

4.3.1 Stage specific effect of HDAC inhibitors on P. falciparum growth in vitro 57

4.3.2 Effects of HDAC inhibitor treatment on P. falciparum morphology 62

4.3.3 Effect of HDAC inhibitors on P. falciparum histone acetylation status 64

4.3.4 Effect of HDAC inhibitors on deacetylase activity of P. falciparum nuclear

extracts 66

4.3.5 HDAC inhibitors alter transcription of alpha-II-tubulin in P. falciparum

parasites 67

4.4 Discussion 68

Chapter 5. Characterisation of P. falciparum histone deacetylase 1 72

5.1 Introduction 73

5.2 Materials and methods 74

5.2.1 Sequence analysis and construction of phylogenetic trees 74

5.2.2 Generation of an in silico PfHDAC1 homology model 74

5.2.3 Generation of PfHDAC1 polyclonal antisera 74

5.2.4 Preparation of RNA and protein for expression profiling 75

5.2.5 Cloning and purification of PfHDAC1 76

5.3 Results 77

5.3.1 Phylogenetic analysis of P. falciparum class I HDAC, PfHDAC1 77

5.3.2 Sequence analysis of PfHDAC1 78

5.3.3 In silico PfHDAC1 homology model 80

5.3.4 Analysis of P. falciparum class II HDACs 81

5.3.5 Stage specific expression of PfHDAC1 during the P. falciparum

intra-erythrocytic developmental cycle 82

5.3.6 Recombinant expression of the PfHDAC1 in E. coli ` 85

5.4 Discussion 90

Chapter 6. Generation and characterisation of PfHDAC1 “disruption” and

“overexpression” mutants 95

6.1 Introduction 96

6.2 Materials and methods 96

6.2.1 Generation of gene disruption constructs 96

6.2.2 Generation of overexpression constructs 97

6.3 Results 98

6.3.1 Characterisation of disruption mutants 98

6.3.2 Generation and analysis of P. falciparum PfHDAC1 overexpression mutants 101

6.3.2.1 Generation of overexpression mutants 101

6.3.2.2 Confirmation of expression of PfHDAC1 102

6.3.2.3 Localisation of PfHDAC1-cmyc and PfHDAC1-GFP fusion protein within

P. falciparum infected erythrocytes 104

6.3.2.4 Comparison of intra-erythrocytic growth of PfHDAC1 overexpression mutants

versus D10 wildtype parasites 107

6.3.2.5 Susceptibility of PfHDAC1 overexpression mutants to antimalarial compounds 110

6.3.2.6 Comparative deacetylase activities of D10 wildtype versus PfHDAC1-cmyc

parasites 111

6.4 Discussion 112

Chapter 7. General discussion and future directions 116

Appendix 118

Supplementary figures 118

Supplementary tables 126

Medias and Buffers 129

List of References 130

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List of Figures

Chapter 1.

Figure 1.1. Schematic representation of the P. falciparum lifecycle, showing examples of antimalarial drugs targeting specific lifecycle stages of the parasite 4

Figure 1.2. Schematic of acetylation and deacetylation of histones by the opposing action of histone acetyltransferases (HATs) and histone deacetylases (HDACs) 9

Figure 1.3. Schematic representation of human versus P. falciaprum class I/II HDAC structures 15

Figure 1.4. Schematic representation of human versus P. falciaprum Sir2 HDAC structures 16

Figure 1.5. General features of hydroxamic acid based HDAC inhibitors 18

Chapter 3.

Figure 3.1. Schematic representation of non-peptidic compounds derived from

L-cysteine and 2-aminosuberic acid 38

Figure 3.2. Oral bioavailability of 2-ASA-14 in BALB/c mice 45

Figure 3.3. In vivo antimalarial efficacy of 2-ASA-9 and 2-ASA-13 in mice 46

Figure 3.4. In vitro antimalarial activity of HDAC inhibitors when combined with known antimalarial drugs 47

Chapter 4.

Figure 4.1. Effect of HDAC inhibitors on the growth P. falciparum over multiple cycles 58

Figure 4.2. Effect of HDAC inhibitors against ring-stage P. falciparum infected erythrocytes growth 59

Figure 4.3. Effect of HDAC inhibitors against trophozoite-stage

P. falciparum-infected erythrocytes growth 59

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Figure 4.4. Stage-specific effect of HDAC inhibitors on P. falciparum ring and trophozoite-stage infected erythrocytes 61

Figure 4.5. Effect of continuous exposure of HDAC inhibitors on growth of P. falciparum ring and trophozoite-stage infected erythrocytes 62

Figure 4.6. Effect of HDAC inhibitor treatment on P. falciparum infected erythrocyte morphology 63

Figure 4.7. Effect of 2-ASA compounds on P. falciparum histones acetylation 65

Figure 4.8. Effect of SAHA and WR301801 on P. falciparum histone acetylation 65

Figure 4.9. Dose dependent inhibition of P. falciparum deacetylase activity by hydroxamate-based HDAC inhibitors 66

Figure 4.10. Effect of HDAC inhibitor treatment on P. falciparumm alpha-II-tubulin gene transcription 68

Chapter 5.

Figure 5.1. Analysis of anti-PfHDAC1 polyclonal antisera 75

Figure 5.2. Phylogenetic relationship of selected class I HDAC homologues 78

Figure 5.3. Multiple sequence alignment of class I HDAC amino acid sequences 79

Figure 5.4. Homology model of PfHDAC1 81

Figure 5.5. Stage-specific transcription profile of Pfhdac1 during the asexual developmental cycle of P. falciparum 83

Figure 5.6. Stage-specific protein expression of PfHDAC1 during the asexual developmental cycle of P. falciparum 84

Figure 5.7. Deacetylase activity in P. falciparum trophozoite nuclear extracts 85

Figure 5.8. Schematic representation of the pRSET-PfHDAC1 prokaryotic expression plasmid (A) and pRIG plasmid (B) 86

Figure 5.9. Pilot expression of recombinant PfHDAC1 (rPfHDAC1-6×His) in BL21

E. coli cells 88

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Figure 5.10. Small scale expression and purification of rPfHDAC1-6×His 89

Figure 5.11. Large scale expression and purification of rPfHDAC1-6×His 90

Chapter 6.

Figure 6.1. Schematic representation of pCHH-Pfhdac1-KO knockout plasmid 99

Figure 6.2. PCR analysis of pCHH-Pfhdac1-KO transfected 3D7 P. falciparum parasites following selection for integrated plasmid 101

Figure 6.3. Schematic representation of PfHDAC1 overexpression plasmid 102

Figure 6.4. Northern blot analysis of PfHDAC1 expression mutants 103

Figure 6.5. Western blot analysis of PfHDAC1 overexpression mutants 104

Figure 6.6. Immunoflourescent assay (IFA) of D10 wildtype and transgenic overexpression parasites 106

Figure 6.7. Live (unfixed) fluorescence images of D10 PfHDAC1-GFP transgenic parasites 107

Figure 6.8. Comparison of overexpression mutants versus wildtype in vitro growth and development 108

Figure 6.9. Differential parasitemia counts of overexpression mutants versus wildtype

Parasites 109

Figure 6.10. Growth inhibition activities of D10 PfHDAC1 transgenic versus

D10 wildtype parasites 110

Figure 6.11. Comparative deacetylase activity in D10 wildtype versus

D10 PfHDAC1-cmyc overexpression parasites 112

Appendix

Figure S1. Multiple sequence alignment of class I HDAC amino acid sequences 118

Figure S2. Multiple sequence alignment of class II HDAC amino acid sequences 122

List of Tables

Chapter 1.

Table 1.1. Classification and key features of human HDACs 11

Table 1.2. Summary of P. falciparum class I/II HDACs 15

Table 1.3. Summary of P. falciparum Sir2 HDACs 17

Table 1.4. Activity of different types of HDAC inhibitors against P. falicapurm versus mammalian cells 21

Chapter 3.

Table 3.1. Comparative toxicities of L-CYS derivatives against P. falciparum versus mammalian cells 43

Table 3.2. Comparative toxicities of 2-ASA derivatives against P. falciparum versus mammalian cells 44

Chapter 4.

Table 4.1. Inhibitory activity of HDAC inhibitors on whole parasites and

P. falciparum deacetylase activity 67

Appendix

Table S1. List of P. falciparum strains 126

Table S2. List of antibodies 126

Table S3. List of primers 127

Table S4. List of hits to C-terminus of PfHDAC1 128

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List of Abbreviations

2-ASA 2-aminosuberic acid ACT Artemisinin combination therapy AFU Arbitrary fluorescence AMA-1 Apical membrane antigen-1 ABHA azelaic bishydroxamic acid BSA Bovine serum albumin

CaCl2 Calcium chloride co-REST corepressor of RE1-silencing transcription factor DMSO Dimethyl sulfoxide DHFR Dihydrofolate reductase DHODH dyhydrooroate dehydrogenase DHPS dihydropteroate synthase ECL Enhanced chemiluminescence EDTA Ethylenediaminetetraacetic acid EGTA Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid FIC Fractional inhibitory concentration GAPDH Glyceraldehyde 3-phosphate dehydrogenase GFP Green fluorescent protein HAT Histone acetyltransferase HCL hydrochloric acid HDAC Histone deacetylase HDLP Histone deacetylase-like protein HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HPLC High performance liquid chromatography HSP70 Heatshock protein 70 IFA Immunofluorescent assay IPTG Isopropyl β-D-1-thiogalactopyranoside KCl Potassium chloride

K2HPO4 Potassium phosphate LB Luria Broth LC-MSMS Liquid chromatography – Tandem mass spectrometry

xvii

L-CYS L-cysteine MEF2 Myocyte enhancer-binding factor 2

MgCl2 Magnesium chloride MSP-1 merozoite surface protein-1 NaCl Sodium chloride

NaH2PO4 Sodium dihydrophosphate NaOH Sodium hydroxide NLS Nuclear localization signal NMT N-myristoyltransferase NuRD Nucleosome localization and deacetylase PBS Phosphate buffer saline PCR Polymerase chain reaction PfCrt P. falciparum chloroquine resistance transporter Pfmdr1 P. falciparum multidrug resistance reporter protein PVDF Polyvinylidene fluoride SDS Sodium dodecylsulfate SDS-PAGE SDS-polyacrylamide gel electrophoresis Sin3 Swi independent 3 Sir Silenced information regulator SMRT Silencing mediator for retinoid and thyroid receptor SAHA Suberoylanilide hydroxamic acid SBHA Suberyl bishydroxamic acid SHA salicylic hydroxamic acid TSA Trichostatin A TEM Transmission electron microscopy WHO World Health Organisation

xviii

Chapter 1

Introduction

Publications arising from data presented in this chapter:

Andrews KT, Tran TN, Wheatley NC, Fairlie DP: Targeting histone deacetylase inhibitors for anti-malarial therapy. Current Topics in Medicinal Chemistry 2009, 9(3):292-308.

Chapter 1

1.1 Malaria – Overview

Malaria remains a leading tropical infectious disease. More than 40% of the world’s population is at risk and over a million malaria-related deaths occur each year, many in children under the age of five in sub-Saharan Africa (WHO, 2008). There are four main species of Plasmodium that are capable of infecting humans, with P. falciparum being the most lethal. P. vivax is also responsible for a significant amount of malaria-related morbidity and predominantly occurs in

South-America and Asian-Pacific regions (Mendis et al., 2001, Price et al., 2007, Baird, 2007).

Clinical symptoms of malaria depend on the seriousness of the disease and can range from fever to severe complications such as renal failure, acidosis, anaemia, organ failure, and coma

(Looareesuwan et al., 1987, Molyneux et al., 1989, Sitprija et al., 1996). If severe malaria is not treated it can lead to cerebral malaria and death. Pregnancy-associated malaria is also a major health concern for women and their unborn children. The World Health Organization (WHO) currently recommends that uncomplicated falciparum malaria be treated with artemisinin-based combination therapies (ACT) which contain artemisinin (or one of its derivatives) together with another antimalarial drug such as lumefantrine, mefloquine, amodiaquine or sulfadoxine- pyrimethamine (SP) (WHO, 2006). However, non-ACT treatment options, including SP and amodiaquine are also recommended in certain regions where ACT is not available (WHO,

2006). Diagnosis and prevention strategies, including vector control, insecticide treated nets, and indoor residual spraying with long-lasting insecticides, are also important in the prevention and management of malaria (WHO, 2008). While a protective and safe vaccine for malaria is needed, this is not currently available (Casares & Richie, 2009). The most promising vaccine currently being assessed in clinical trials is RTS,S. RTS, S is a subunit vaccine that uses the hepatitis B surface antigen as a carrier for epitopes derived from circumsporozoite protein of P. falciparum (Garcon et al., 2003). Recent data have demonstrated that while protection is modest, that this vaccine can protect children against clinical (35%) and severe malaria (48%)

(Alonso et al., 2005).

Chapter 1

1.2 Plasmodium lifecycle

Plasmodium parasites have a complex lifecycle that involves a definitive host, the female

Anopheles mosquito where sexual reproduction occurs, and the human host where asexual reproduction occurs (Figure 1.1). The lifecycle typically begins when a female Anopheline mosquito ingests the sexual stage gametocytes that circulate in the peripheral blood of the human host during a blood meal (Figure 1.1A). Fertilization between the male and female gametocytes takes place in the mosquito mid-gut (Figure 1.1B), followed by maturation of zygotes to ookinetes. Ookinetes cross the mosquito mid-gut and mature to form oocysts containing infective sporozoites. Sporozoites are then trafficked to the salivary glands of the infected mosquito where they are injected into another human host during the next blood meal

(Figure 1.1C). Within the host, sporozoites travel to the liver where they infect hepatocytes and mature into tissue schizonts containing thousands of infective merozoites (Figure 1.1D; (Sinnis

& Coppi, 2007)). Upon rupture of the tissue schizonts, merozoites are released and travel to the blood stream where they can invade erythrocytes and undergo asexual reproduction. In P. vivax, sporozoites are also able to remain dormant in liver cells (termed hyponozoites). Asexual erythrocytic stages develop into rings, trophozoites, and schizonts. Mature schizonts contain up to 32 daughter merozoites (Figure 1.1E; see (Bannister et al., 2000) and references therein) which upon rupture can invade additional host red blood cells. This asexual blood phase of the parasite lifecycle in the human host lasts about 48 h (72 h in P. malariae) and it is during this phase that the clinical symptoms of malaria can occur. It is also during the asexual reproduction phase that some asexual parasites can, via a process that is incompletely understood, undergo transformation into male and female gametocytes that may be taken up by a mosquito during a blood meal, thereby completing the parasite’s lifecycle.

Chapter 1

Figure 1.1. Schematic representation of the P. falciparum lifecycle, showing examples of antimalarial drugs targeting specific lifecycle stages of the parasite. (A) The female Anopheline mosquito takes a blood meal; (B) sexual reproduction takes place in the mosquito mid-gut; (C) infectious sporozoites are injected into the human host during a blood meal and travel to the liver; (D) sporozoites infect hepatocytes and replicate followed by release of merozoites into the host’s blood stream, and (E) asexual reproduction occurs in host erythrocytes (intra-erythrocytic cycle), during which time gametocytes may be formed and taken up by a feeding female Anopheline mosquito, completing the lifecycle (Andrews et al., 2009).

Key stages in the malaria parasite’s lifecycle are targets for current and potential antimalarial intervention and treatment strategies. Antimalarial drugs that target different lifecycle stages include: (i) proguanil (usually administered in combination with atovaquone) which kills sporozoites (sporontocidal agent), and tissue schizonts (tissue schizontocide) (ii) primaquine and tafenoquine which act on tissue schizonts (tissue schizontocides) together with sexual forms in the blood stream of human hosts (gametocytocidal agents) and (iii) quinine, mefloquine, and chloroquine (Figure 1.1) which target the asexual erythrocytic stages of parasite development.

Most of the antimalarial agents currently in use target the asexual stage malaria parasites. 4

Chapter 1

1.3 Malaria parasite drug resistance

The spread of drug resistant parasites in most malaria endemic regions throughout the world is a major concern and is a particular problem in P. falciparum. P. falciparum parasites resistant to nearly all known antimalarial drugs have been reported. There have also been recent reports describing parasite populations resistant to ACTs on the Thai-Cambodia border (discussed below in more detail; (Noedl et al., 2008, Dondorp et al., 2009, Wongsrichanalai & Meshnick,

2008). Despite the declining efficacy of chloroquine against P. falciparum, chloroquine (and chloroquine combined with primaquine) is still used to treat P. vivax infections (Yeshiwondim et al., 2009).

One of the major breakthroughs since the discovery of chloroquine was the discovery of artemisinin and its potent antimalarial activity. Various artemisinin derivatives are now available (eg. artemether, artesunate, and dihydroartemisinin). Artemisinin is derived from

“qinghao” or sweet wormwood (Yu & Zhong, 2002), a plant that has traditionally been used by the Chinese to treat malaria. Artemisinin-based combination therapy (ACT) is now the recommended treatment for uncomplicated malaria (WHO, 2006). The rationale for the use of two drugs that act on independent parasite targets is that it may help reduce the likelihood of selecting for parasite resistance. Unfortunately recent reports indicate that even ACT efficacy may be under threat (Dondorp et al., 2009). The emergence of ACT resistance in the Thai-

Cambodia border is due to many possible factors. For example, unreliable services and poor diagnostics at public health facilities is thought to be partially responsible for patient refusal to seek treatment from the public health sector (Wongsrichanalai & Meshnick, 2008). Other reasons include the short half life of artesunate compared to mefloquine suggests tolerance to mefloquine could develop when treated patients are reinfected (Hastings & Ward, 2005).

Parasites adopt a number of different mechanisms to become drug resistant. Resistance to chloroquine, a once very cheap and safe antimalarial agent, for example, has been associated with mutations in two different transport genes P. falciparum chloroquine resistance transporter

Chapter 1

(Pfcrt) and P. falciparum multidrug resistance reporter protein (Pfmdr1) (Duraisingh &

Cowman, 2005, Cooper et al., 2005). Additional mutations have also been associated with drug resistant parasites. Resistance to atovaquone can be mediated by a single point mutation in the cytochrome b gene (Musset et al., 2006). Likewise, point mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes can result in pyrimethamine and sulfadoxine resistant parasite populations (Ndiaye et al., 2005). The development of resistance to these antimalarial agents is fuelled by their long in vivo half lives which can result in parasites being exposed to prolonged sub-lethal concentrations of each individual drug (Watkins

& Mosobo, 1993). SP also induces gametocytogenesis resulting in increase gametocyte density, and hence might increase transmission rates (Bousema et al., 2003).

The possible failure of current ACT regimens over the coming years underscores the need for developing new antimalarial drugs and drug combinations. In addition to efficacy against drug resistance parasites, new drugs need to be cheap, orally bioavailable, safe for children and pregnant women, and effective in killing parasites in a period of time that ensures good clinical compliance (treatments of 3 days or less). One of the major aims in reducing the spread of antimalarial resistance is to limit the density of both asexual and sexual stage parasites within the host, which will ultimately help to reduce transmission of the parasite to the mosquito vector. Therefore, drugs that can stop asexual stage parasites before they can undergo gametocytogenesis, drugs that kill or arrest gametocytes, or drugs that reduce gametocyte loads are highly desirable. In 2007, the Bill and Melinda Gates foundation launched a US$20 million initiative to fund the establishment of the Worldwide Antimalarial Resistance Network

(http://www.wwarn.org/) which aims to better manage the global issue of drug resistance and eventually to eradicate malaria.

1.4 Approaches to drug discovery for malaria

The high burden of malaria, the lack of a licensed vaccine, and the global problem of parasite drug resistance is driving the need to discover new chemotherapeutics that (1) act on different

Chapter 1 parasite targets to existing drugs, (2) have complimentary pharmacokinetic profiles, and (3) are cost effective (Nwaka & Ridley, 2003, Trouiller et al., 2002). Broadly, three major strategies can be used to identify new lead antimalarial compounds, including “label extension”, “de novo”, and “piggy-back” approaches (Nwaka & Hudson, 2006).

The “label extension” approach has historically been used to identify new drugs for malaria and other tropical diseases, based on drugs that have previously been used to treat other human diseases (Pink et al., 2005, Rosenthal, 2003, Witty, 1999). Advantages of this method are reductions in cost and time to market. Examples include folate antagonists such as tetracycline, doxycycline and other antibiotics which were developed for their antibacterial properties, but were also found to have antimalarial activity (Dahl et al., 2006). Although atovaquone was initially identified as an antimalarial compound it was only after it was developed to treat

Pneumocystis that this compound was re-evaluated for use against malaria parasites (Madden et al., 2007). Atovaquone has synergistic antimalarial activity when combined with proguanil

(Canfield et al., 1995) and this combination, marketed as Malarone, is approved for treatment and chemoprophylaxis of malaria

“De novo” drug discovery is a highly attractive strategy to identify novel anti-parasitic compounds due to the large scale capability of high-throughput (HTS) or medium throughput

(MTS) screening programs. One example of this approach is the recent development of a DAPI

P. falciparum growth assay adapted to 384-well plate format that was used to screen ~79,000 compounds from the Institute of Chemical and Cell Biology – Longwood libraries (Baniecki et al., 2007). Of these, 900 compounds demonstrated 90% growth inhibition of P. falciparum infected erythrocytes at 30µM. Despite recent success of screening programs, this approach has produced only a few successful leads so far (Bleicher et al., 2003, Gribbon & Sewing, 2005).

Lead discovery process is also costly and inheritably risky with an estimated cost of greater

US$300 million to bring a new antimalarial to market and it has been estimated that the failure rate in phase two clinical trials for a new antimalarial is up to 50% (Pink et al., 2005). In order

Chapter 1 for this approach to continue to be successful, ongoing public-private partnership and funding will be required.

Another approach is “piggy-back” drug discovery which exploits chemical leads being commercially developed for use in one disease for which the molecular target is present in another. An example of the “piggy-back” approach is the investigation of P. falciparum N- myristoyltransferase (NMT) enzymes which catalyse the transfer of the fatty acid myristate from myristoyl-CoA to the N-terminal of glycine of target eukaryotic, bacterial or viral proteins

(Maurer-Stroh et al., 2002, Maurer-Stroh & Eisenhaber, 2004). NMT is also been investigated as a potential drug target in kinetoplastid parasites (Gelb et al., 2003) as well as a novel cancer therapy (Selvakumar et al., 2002). Another example of this approach is histone deacetylase

(HDAC) enzymes that are being targeted for cancer therapy and using them as new antimalarial targets (Andrews et al., 2000, Darkin-Rattray et al., 1996). As discussed below, P. falciparum

HDACs and their potential as new drug targets in malaria parasites are the focus of this thesis.

1.5 P. falciparum HDACs as potential new drug targets:

The first evidence to suggest that Plasmodium HDACs may represent new antimalarial drug targets came from a study which showed the natural product HDAC inhibitor apicidin was active against P. falciparum in vitro and against the lethal rodent malaria P. berghei in vivo

(Darkin-Rattray et al., 1996). As in mammalian cells, apicidin was shown to cause hyperacetylation of P. falcipaurm histones, indicating that this compound was acting as a

HDAC inhibitor in the parasite. A P. falciparum protein with homology to eukaryotic class I

HDACs has since been identified and has been suggested as a putative enzyme target of HDAC inhibitors in the parasite (Joshi et al., 1999). The general features of eukaryotic HDACs, and the

HDACs that have been identified in P. falciparum, are discussed in the following sections.

Chapter 1

1.6 HDACs: function and classification

Chromatin is made of repeating units of nucleosomes consisting of an octamer of core histones

(two each of H2A, H2B, H3, and H4) to which ~150 base pair DNA segments are wrapped in

1.75 superhelical turns like “beads on a string” (Figure 1.2). Core histones are amongst the most abundantly expressed eukaryotic proteins. The structure of each histone consists of a C- terminal tail, a central “histone-fold” domain and a flexible N-terminal tail that is subjected to different post-translational modifications such as ADP-ribosylation, ubiquitination, phosphorylation, methylation and acetylation. Together with other enzymes HDACs and histone acetyltransferases (HATs) are key histone modifying enzymes that are involved in “writing” and

“reading” the “histone code”. The “histone code” has been defined as distinct modifications on one or more tails of histones that act sequentially or together to facilitate downstream events

(Strahl & Allis, 2000). Histone modifying enzymes are crucial for modulating cell chromatin structure and gene expression in higher eukaryotic organisms (de Ruijter et al., 2003). Inhibition of histone acetylation or deacetylation can both activate and suppress transcription (Van Lint et al., 1996, Mitsiades et al., 2004). HDAC inhibitors have been widely evaluated for their cytotoxic, anticancer activity and other potential therapeutic properties (For recent reviews see

Gallinari et al., 2007, Rasheed et al., 2007). HAT inhibitors are also being investigated for therapeutic use (Balasubramanyam et al., 2003, Balasubramanyam et al., 2004), although are less well studied than HDAC inhibitors.

Figure 1.2. Schematic of acetylation and deacetylation of histones by the opposing action of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Repeating nucleosomal units composed of core histones (orange) are shown with wrapped DNA (green).

Chapter 1

1.7 Classification and function of eukaryotic HDACs

HDACs are members of a large family of proteins which can be divided into four classes based on sequence similarity, subcellular localization, and co-factor dependency. The human genome encodes 11 zinc-dependent HDAC family members (Table 1.1) which includes HDAC1, 2, 3, and 8 (class I HDACs) and HDAC4, 5, 6, 7, 9, and 10 (class II HDACs) (reviewed in de Ruijter et al., 2003). HDAC11, which displays similar gene features to class I HDACs but also has similarity to bacterial deacetylase-like protein, has been recently consigned as a separate class

(class IV) (Gao et al., 2002). HDAC11 does not appear to associate with class I containing multi-protein complexes but has been shown to associate with the class II protein, HDAC6 (Gao et al., 2002). The human class III family of HDACs, or sirtuin proteins (Silenced information regulator (Sir)) comprises SirT1-7 (reviewed in Dali-Youcef et al., 2007). Class III HDACs share no sequence similarity with either class I or II HDACs and these enzymes differ in their co-factor requirement, using nicotinamide adenosine dinucleotide (NAD) not Zn2+ as co-factor

(Dali-Youcef et al., 2007).

1.7.1 Key features of class I HDACs

Class I HDACs share a similar zinc-dependent deacetylase domain but are smaller than class II

HDACs and localize exclusively to the nucleus (de Ruijter et al., 2003) (Table 1.1). The catalytic core domain located in the N-terminal region forms a major part of these enzymes

(>50% of total protein). These enzymes are known to deacetylate histone proteins (Li et al.,

2004), but also non-histone proteins such as the transcription factor p53 (Luo et al., 2000). In vivo, class I HDACs form multi-protein complexes consisting of a variety of proteins, including other HDACs. Proteins in these complexes include those necessary to modulate the activity of the complex and those needed to recruit HDACs to the promoters of genes (Zhang et al., 1999).

In mammalian cells HDAC1 and HDAC2 are known to associate together (Taplick et al., 2001) in numerous multi-protein complexes including the Swi independent 3 complex (Sin3) (Heinzel et al., 1997), the nucleosome localization and deacetylating complex (NuRD) (Zhang et al.,

1999) and the corepressor of RE1-silencing transcription factor (co-REST) complex (You et al.,

Chapter 1

2001). Although HDAC3 shares a similar deacetylase domain to other class I HDACs, it associates with different multi-protein complexes containing class II HDACs (Fischle et al.,

2001, Yang et al., 2002). These observations indicate that members of this class of HDACs are not redundant in function and may demonstrate enzyme-specific activities (at least in higher eukaryotes).

Table 1.1. Classification and key features of human HDACs. Class HDAC Characteristics I 1,2 3, 8a  Homology to yeast Rpd3  ~390 amino acid (aa) catalytic domain (Zn2+- dependent activity)  42-55 kDa  Sensitive to TSA, SAHA and other HDAC inhibitors  Ubiquitously expressed/ Mainly nuclear localization

IIa 4, 5, 7, 9a  Similarity to Had 1  ~390 aa catalytic domain  Zn2+-dependent activity  120-130 kDa

a IIb 6 &10  HDAC6 – 2 deacetylase domains  Sensitive to TSA, SAHA and other HDAC inhibitors  Interact with different set of proteins other than class I  Tissue specific expression/nuclear or cytoplasmic localization

III SIRT1-7b  Sir2 – founding member (yeast) (sirtuin)  conserved 275 aa catalytic domain unrelated to class I & II HDACs (NAD+ required as a co-substrate)  silence transcriptional regulators/other cellular processes  SIRT1 – deacetylates p53  Some nuclear, some cytoplasmic

IV 11c  Tissue specific expression/nuclear localization  Associated with HDAC6 in vivo a See reference (de Ruijter et al., 2003) b See reference (Dali-Youcef et al., 2007) c See reference (Gao et al., 2002)

Chapter 1

1.7.2 Key features of class II HDACs

In humans, class II HDAC members are sub-grouped as either class IIa (HDAC4, HDAC5,

HDAC 7, and HDAC9) or class IIb (HDAC6 and 10) enzymes (Hubbert et al., 2002). A major difference between class IIa and class IIb proteins is that HDAC6 contains two tandem HDAC domains, and HDAC10 has an additional partial HDAC domain (Guardiola & Yao, 2002). Class

IIa HDACs can be localized in the nucleus and cytoplasm of cells (Grozinger & Schreiber,

2000, Wang et al., 2000). HDAC6 is predominantly localized to the cytoplasm and specifically deacetylates alpha-tubulin (Hubbert et al., 2002). Class II HDAC members all possess a similar catalytic deacetylase domain (amino acid sequence identity 39 – 74%), but a less conserved N- terminal sequence (amino acid identity 40 – 42%) (Bertos et al., 2001). Class II members also contain conserved protein-binding motifs which are important for their function and regulation

(Bertos et al., 2001). In the nucleus, class IIa HDACs interact with transcription factors

(Lemercier et al., 2000) and co-repressors (Huynh et al., 2000) to facilitate the repression or silencing of gene transcription. The N-terminal region of class IIa HDACs contain phospho- serine or phospho-threonine motifs that are recognised by the protein 14-3-3 (that has no catalytic activity) which causes the cytoplasmic localisation of these HDACs (Grozinger &

Schreiber, 2000) suggesting subcellular localisation may have an important role in controlling the co-repression function of these HDACs. Unlike class I HDACs, class II HDACs display tissue-specific expression in mammals (Grozinger et al., 1999) indicating these enzymes are also not redundant in function. The transcription factor myocyte enhancer-binding factor 2

(MEF2) is known to interact with HDAC4 and HDAC5 (Lu et al., 2000). The MEF2-HDAC complex exerts transcriptional repression through the deacetylase activity of the HDAC enzymes at the local chromatin level (Lu et al., 2000). Class IIa HDACs also interact in a multi- protein complex with the co-repressor silencing mediator for retinoid and thyroid receptor

(SMRT) in transcriptional repression (Huang et al., 2000). SMRT interaction with HDAC3 is known to be important for HDAC 3 deacetylase activity in transcriptional regulation (Guenther et al., 2001). While, class II HDACs including HDAC4, HDAC5 and HDAC7 also interact with

Chapter 1

SMRT in multi-protein complexes (Fischle et al., 2002), their roles in transcriptional regulation remains unknown.

1.7.3 Key features of class III (Sir2) HDACs

Class III HDACs (also referred to as Sir2 proteins) differ from class I and II HDACs in that they require NAD+ as co-factor for deacetylation, catalysing the acetylated lysine substrate to a deacetylated lysine product and the by-products nicotinamide (an inhibitor of sirtuins) and O- acetyl-ADP-ribose (Marmorstein, 2004). In addition, Sir2 proteins also possess ADP-ribosylase activity and in yeast ScSir2 can ADP-ribosylate itself and its histone substrates (Tanny et al.,

1999). ScSir2 is an important protein in epigenetic transcriptional control in which the Sir2 protein interacts with other Sir proteins (Sir3 and Sir4) to form the Sir2/3/4 complex. This complex has been shown to associate with repressed genes in sub-telemeric regions (Strahl-

Bolsinger et al., 1997). In humans the seven members of the Sir2 family display tissue-specific expression and different sub-cellular localization. These Sir2 proteins are associated with many functions including gene silencing, transcriptional regulation, longevity and calorie restriction

(Dali-Youcef et al., 2007) and hence they represent attractive targets for therapeutic interventions.

1.8.1 P. falciparum class I and II HDACs

The P. falciparum genome (Gardner et al., 2002) contains at least five putative HDAC- encoding genes including one class I homologue of the yeast Rpd3 protein (PFI1260c), two class II homologues of the yeast HDA1 protein (PF14_0690 and PF10_0078) (Table 1.2), and two class III (Sir2) homologues of the yeast Hst1 protein (PF13_0152 and PF14_0489).

The amino acid sequence of the class I HDAC homologue, PfHDAC1, contains a conserved class I/II deacetylase domain of ~300 amino acids at the amino terminus, and a carboxy- terminal extension with no homology with known class I HDACs (Figure 1.3). Localization of

PfHDAC1 in the parasite nucleus was first reported almost a decade ago (Joshi et al., 1999) and

Chapter 1 it has been proposed as the most likely target of HDAC inhibitors which are under development as new antimalarial agents (Darkin-Rattray et al., 1996). PfHDAC1 transcript is found during the late stages of the parasite asexual cycle (trophozoite and schizonts) and in gametocytes

(Joshi et al., 1999). More recently, proteome wide analysis of P. falciparum parasites has shown that PfHDAC1 is present in asexual stages merozoites, trophozoite and schizont stages (Florens et al., 2004, Florens et al., 2002). More recently, recombinant PfHDAC1 has been expressed in insect cells (Patel et al., 2009) and has been shown to deacetylate lysine peptide substrates, however its activity on free histones and nucleosomal histones has not yet been investigated.

The activity of recombinant PfHDAC1 is inhibited by HDAC inhibitors such as trichostatin A and apicidin, but is insensitive to the Class III (Sir2) inhibitor splitomicin (Patel et al., 2009).

Almost nothing is known about the role of this protein in P. falciparum including whether it is essential to the parasite and what other proteins it interacts with. Likewise, the mechanism(s) that regulate PfHDAC1 activity in the parasite are unknown.

The P. falcipaurm genome also contains two putative class II HDACs, termed PfHDAC2 and

PfHDAC3, on chromosome 14 and chromosome 10, respectively (Table 1.2). Both of these

HDAC homologues are large proteins and have very limited sequence homology with other class II proteins, except in the deacetylase domain in which conserved deacetylase signature motifs are found. Currently, there is very limited data available on the class II HDACs in P. falciparum. Mass spectrometry data suggest that PfHDAC2 is expressed in trophozoites and gametocytes. PfHDAC3 appears to have gametocyte specific expression (Florens et al., 2002).

The extremely long sequences of PfHDAC2 (2251aa) and PfHDAC3 (2379aa) coupled with the

A-T rich genome of P. falciparum make research with these genes difficult. However, it will be interesting to determine if these proteins are essential to the parasite or play a role in the transition between different developmental stages in the parasite.

Chapter 1

Table 1.2. Summary of P. falciparum class I/II HDACs. Name Molecular Class Features/Additoinal Informationb, c, d, e [gene ID]a Weight PfHDAC1f Class I 51  Transcribed in T, S, and G [PFI1260c]  Expressed in T, S, M, IE  ~50 kDa protein detected in Triton-X insoluble infected erythrocyte extracts  Predominant nuclear localization  Lack of functional HDAC activity of recombinant protein expressed in E. coli (amino acids 16-449)  No Y2H interactions detected

PfHDAC2 Class I/II 269  Transcribed in R, T, S, M, S [PF14_0690]  Expressed in G and T  Conserved domains: Ankyrin repeat; HDAC  Identified Y2H interactions: S-adenosyl-L- homocysteine hydrolase (PFE1050w)

PfHDAC3; Class I/II 282  Transcribed in intraerythrocytic stages and [PF10_0078] G  Expressed in G  Y2H interactions: knob associated histidine rich protein (KAHRP; PFB0100c) aP. falciparum, gene identification numbers Gene ID from (Aurrecoechea et al., 2009) bP. falciparum transcription data obtained from (www.PlasmoDB.org)(Bozdech et al., 2003, Le Roch et al., 2003) cP. falciparum mass spectrometry based evidence available on (www.PlasmoDB.org). M, merozoite; T, trophozoite, S, schizont; G, gametocyte; Sp, sporozoite) (Florens et al., 2002) dConserved domains: conserved domains identified by BLASTp against the human genome are indicated (Altschul et al., 1990) e Y2H interaction (LaCount et al., 2005) f (Joshi et al., 1999) MW; molecular weight (kDa)

100 aa

Figure 1.3. Schematic representation of human versus P. falciaprum class I/II HDAC structures. (A) Class I HDAC homologues from human (HsHDAC1) and P. falciparum (PfHDAC1). (B) Class II HDAC homologues from human (HsHDAC4) and P. falciparum (PfHDAC2 and PfHDAC3). Core deacetylase catalytic domains are shown as red striped shading.

Chapter 1

1.8.2 P. falciparum Sir2 HDACs

The P. falciparum genome contains two Sir2 homologues (Table 1.3; Figure 1.4). The PfSir2A gene encodes for a 273 amino acid protein with some sequence conservation in the deacetylase domain to human SirT2 (23%) and Archeoglobus fulgidus Af1 (35%). PfSir2A is closely related to the “type III” sirtuin family (Merrick & Duraisingh, 2007). Like other Sir2 proteins, the

PfSir2A acts as a lysine histone deacetylase that can ADP-ribosylate itself and histones

(Merrick & Duraisingh, 2007). In addition, PfSir2A has been found to associate with the P. falciparum sumoylation protein PfSUMO suggesting this protein might be subject to posttranslational modification (Issar et al., 2008). PfSir2B is a much larger protein which contains a deacetylase domain with greatest similarity to “type IV” sirtuins (Tonkin et al.,

2009). Both PfSir2s have been individually genetically disrupted in the intra-erythrocytic developmental cycle of P. falciparum indicating that this class of HDAC is not essential to the parasite development (at least in vitro) limiting their suitability as drug targets (Duraisingh et al., 2005, Tonkin et al., 2009). However, these HDACs have been shown to play an important role in epigenetic regulation of P. falcipaurm var genes which are involved in pathogenesis

(cytoadherence and antigenic variation) (Tonkin et al., 2009, Duraisingh et al., 2005, Freitas-

Junior et al., 2005). A recent study by Tonkin et al., (2009) suggests these two PfSir2s may be acting in a cooperative manner in the transcriptional regulation of individual var genes. Future studies in which disruption of both PfSir2s in P. falciparum may provide new insights into the roles of these HDACs in P. falciparum.

HsSirT2

PfSir2A PfSir2B

100 aa

Figure 1.4 Schematic representation of human versus P. falciaprum Sir2 HDAC structures. Shown are human (HsSirT2) and P. falciparum (PfSir2A and PfSir2B). The core deacetylase catalytic domains are shown as red.

Chapter 1

Table 1.3. Summary of P. falciparum Sir2 HDACs. Name Molecular Class Features/Additoinal Informationb, c, d, e [gene ID]a Weight  Transcripts in R, T, S, G, S PfSir2A; Sir2 30  Expression in all asexual stages f, g [PF13_0152]  Nuclear pole/punctate perinuclear localization  Co-localization with heterochromatin and telomeric clusters  HDAC and ADP-ribosyltransferase activity  Inhibited by nicotinamide  Not essential in vitro  Associated with silencing of some var and rif genes  Y2H interactions : none detected

PfSir2B; Sir2 155  Expressed in Sp [PF14_0489]h  Not essential in vitro  Associated with silencing of some var genes aP. falciparum, gene identification numbers Gene ID from (Aurrecoechea et al., 2009) bP. falciparum transcription data obtained from (www.PlasmoDB.org)(Bozdech et al., 2003, Le Roch et al., 2003) cP. falciparum mass Spectrometry based evidence available on (www.PlasmoDB.org). M, merozoite; T, trophozoite, S, schizont; G, gametocyte; Sp, sporozoite) (Florens et al., 2002) dConserved domains: conserved domains identified by BLASTp against the human genome are indicated (Altschul et al., 1990) e Y2H interaction (LaCount et al., 2005) f (Duraisingh et al., 2005) g (Merrick & Duraisingh, 2007) h (Tonkin et al., 2009) MW; molecular weight (kDa)

1.9 HDACs as anti-cancer drug targets

Human HDACs have been recognised drug targets for decades and inhibitors of these enzymes have been developed as both new anticancer agents and as tools to study the function of acetylation modification on chromatin structure and gene expression. Altered expression of genes involved in cellular differentiation, cell cycle, and apoptosis can lead to aberrant transcription and induce cancer (Mai et al., 2005). Some HDAC inhibitors have been shown to inhibit tumour growth including a metabolite of Streptomyces hygroscopicus, trichostatin A

(TSA), which was one of the first HDAC inhibitors identified (Tsuji et al., 1976). TSA is a linear compound consisting of a terminal hydrophobic group, an aliphatic hydrocarbon spacer, and a hydroxamic acid zinc binding group (Figure 1.5).

Chapter 1

Figure 1.5. General features of hydroxamic acid based HDAC inhibitors. The general features of hydroxamate HDAC inhibitors include a “cap region” that blocks the entrance to the active site, an aliphatic “linker region” that spans the length of the hydrophobic binding tunnel of the active site, and the hydroxamic acid group that chelates zinc (zinc binding group) (Drummond et al., 2005). The chemical structure of TSA is shown as an example.

The hydroxamic acid group of TSA has been shown to inhibit class I HDACs by binding to the enzyme catalytic pocket (Finnin et al., 1999, Somoza et al., 2004). While TSA has been shown to have potent anti-proliferative activity against tumour cells (Suzuki et al., 2000), its in vivo activity has been less promising due to its rapid metabolic degradation (Elaut et al., 2002).

However, the chemical structure of TSA has become the basic scaffold of new generation of synthetic hydroxamic acid based analogues. Some of the first generation analogues display selective anti-tumour activity inducing cellular differentiation, growth arrest and apoptosis in various types of tumour cells with minimal effect on normal cells (Marks et al., 2001).

SAHA was the first compound from the hydroxamic acid class of HDAC inhibitors to receive clinical approval for the treatment of advanced cutaneous T-cell lymphoma (Grant et al., 2007).

This compound is a potent inhibitor of class I/II HDACs (human HDAC1, 2, 3 and 6) (Richon et al., 1998) but the anti-tumour mechanism of this drug is complex and not yet fully understood. SAHA causes the accumulation of acetylated histones in both tumour and cancer cells (Ungerstedt et al., 2005) and at concentrations that induce apoptosis (programmed cell death), SAHA up-regulates the expression of the cyclin dependent kinase (cdk) inhibitor p21WAF1 (Zhang et al., 2005). In contrast, other genes are repressed by SAHA treatment

Chapter 1 including cyclin D1, ErbB2, thymidylate synthase and importin b (Glaser et al., 2003). In addition to SAHA’s activity on histones, this compound also causes acetylation of numerous non-histone proteins including the transcription factor p53, alpha-tubulin and the heat-shock protein Hsp90 (Marks et al., 2001, Bali et al., 2005). While, the mechanism of action of SAHA is multifaceted, this compound has low toxicity in clinical studies (Grant et al., 2007).

1.10 HDAC inhibitors as antimalarial agents

It has been over a decade since the first study demonstrating the broad spectrum antiprotozoal activity of the natural product derived cyclic tetrapeptide HDAC inhibitor, apicidin (Table 1.4).

This compound displays nanomolar antiproliferative activities in vitro against P. falciparum intra-erythrocytic stages (IC50 200nM), T. gondii tachyzoites (IC50 13nM), and C. parvum (IC50

48nM) (Darkin-Rattray et al., 1996) (Table 1.4). Like other HDAC inhibitors, apicidin causes accumulation of acetylated histones in P. falciparum (Darkin-Rattray et al., 1996). Furthermore, apicidin also inhibits deacetylase activity in E. tenella (coccidian apicomplexan parasite) extracts at low nanomolar concentrations (IC50 0.7nM) indicating this compound may be inhibiting a HDAC enzyme in apicomplexan parasites (Darkin-Rattray et al., 1996). Apicidin also demonstrated oral antimalarial activity in mice infected with P. berghei but no cure was obtained (Darkin-Rattray et al., 1996). Unfortunately apicidin also kills mammalian cells at similar concentrations (IC50 50 – 100nM). It is also poorly bioavailable (Darkin-Rattray et al.,

1996, Meinke et al., 2000). The activity of apicidin on histone acetylation is likely due to the similarity in structure of the Aoda (2-amino-8-oxodecanoic acid) side chain and the acetylated lysine of histone substrate (Meinke et al., 2000) which may contribute to the general toxicity of this inhibitor. Extensive structure activity relationship (SAR) studies have been carried out to evaluate the biological contribution of the amino acid residues of apicidin (Meinke et al., 2000,

Colletti et al., 2001b, Colletti et al., 2001a). However, these studies have not been able to generate derivatives with selective antimalarial activity.

Chapter 1

In terms of antimalarial potential, HDAC inhibitors bearing a hydroxamic acid zinc binding group have been the most extensively studied. TSA was the first hydroxamate based HDAC inhibitor to be found to have activity against P. falciparum in vitro (Darkin-Rattray et al., 1996).

This compound has nanomolar antiproliferative activity against the multi-drug resistant P. falciparum Dd2 strain (IC50 8nM) and drug sensitive 3D7 strain (IC50 11nM) (Andrews et al.,

2000) (Table 1.4). Like apicidin, TSA has poor bioavailability and is toxic in vivo (Elaut et al.,

2002, Fenic et al., 2004).

Previous studies with SAHA have shown this HDAC inhibitor is moderately active against P. falciparum in vitro (IC50 0.94 – 1.78 µM) (Mai et al., 2004). However, more recent work has shown that SAHA may have better in vitro antimalarial activity than previously expected (IC50

~100nM) and thus suggests this HDAC inhibitor may warrant further investigation (Dow et al.,

2008). The experimental hydroxamate suberyl bishydroxamic acid (SBHA) also has similar in vitro activity to SAHA against P. falciparum but it is more selective in killing parasite over mammalian cells (IC50 0.8 – 1.3µM) (Andrews et al., 2000). Furthermore, this compound has shown to delay the onset, and control, the parasitemia of mice infected with P. berghei

(Andrews et al., 2000).

Chapter 1

Table 1.4. Activity of different types of HDAC inhibitors against P. falicapurm versus mammalian cells. IC50 (µM) Mammalian Selective Compound Structure P. falciparum cells Indexh Cyclic peptide

a Apicidin 0.2 0.05 – 0.1 <1

Hydroxamates O O OH N b H TSA N 0.008 – 0.011 0.2 18 – 25

cSAHA 0.1 – 1.78 >20 11 – 200

O H N OH d HO N H SBHA O 0.8 – 1.3 50 – 300 38 – 375

e WR301801 0.003 0.6 600

O H N N H N N H f O 2-ASA-14 CH2 0.013 – 0.033 0.34 – 0.95 10 – 73

HN OH Benzamides O O N NH 2 H H N g N MS-275 O 7.8 – 8.3 0.04 – 4.71 <1 a IC50 against P. falciparum and HeLa cells (Darkin-Rattray et al., 1996) b IC50 against P. falciparum (Andrews et al., 2000) and IC50 against neonatal foreskin fibroblast cells (NFF) (Glenn et al., 2004) c IC50 against P. falciparum (Mai et al., 2004, Dow et al., 2008) and IC50 against mammalian cells (Fedier et al., 2007) d IC50 against P. falciparum (Andrews et al., 2000) and IC50 against human dermal fibroblast cells (HDF) (Brinkmann et al., 2001) e IC50 against P. falciparum and mammalian cells (Dow et al., 2008) f IC50 against P. falciparum (Andrews et al., 2008) and IC50 against NFF (Glenn et al., 2004) g IC50 against P. falciparum (Andrews et al., 2008) and IC50 against mammalian cells (Saito et al., 1999) h Selective index was calculated as IC50 values of mammalian cells/IC50 values of P. falciparum

Chapter 1

A panel of ~50 new hydroxamate compounds derived from phenylthiazolyl that inhibit eukaryotic HDACs, have recently been screened for activity against P. falciparum in vitro (Dow et al., 2008). These compounds display picomolar to nanomolar activity against various P. falciparum strains. Furthermore, some structure activity relationship was observed with the most active compounds in the series containing amino or hydroxyl substitution in the meta- position of the phenyl ring in the cap region (Table 1.4; WR301801). One of the most potent compounds in this series, WR301801 (IC50 0.6 – 1.8nM against P. falciparum in vitro) (Dow et al., 2008), was also up to 600 times more selective in killing P. falciparum parasites over mammalian cells. While the mechanism of antimalarial activity has not been confirmed data suggest that WR301801 is acting as a HDAC inhibitor. This compound hyperacetylates P. falciparum histones and inhibits parasite deacetylase activity (Dow et al., 2008) (see chapter

5.3.2). WR301801 is orally bioavailable but is rapidly metabolised by mouse and human liver microsomes with a proposed mechanism of metabolism being hydrolysis of the hydroxamate group (Dow et al., 2008). The rapid metabolisim of this compound may be associated with its failure to produce cures as a monotherapy in a lethal mouse malaria model and in an Aotus monkey model infected with P. falciparum strain FVO parasites (Dow et al., 2008). Cures were, however, able to be obtained in mice when 54mg/kg of WR301801 was combined with

64mg/kg chloroquine (Dow et al., 2008), or when the compound was used in a prophylactic rather than treatment mode (Agbor-Enoh et al., 2009).

Few non-hydroxamate-bearing HDAC inhibitors have been investigated for antimalarial activity. Benzamides, including MS-275 have undergone phase II clinical trial for the treatment of melanoma (Hauschild et al., 2008). However MS-275 displays only moderate activity against

P. falciparum in vitro (IC50 7.8 – 8.3µM). A recent cell-based optimization study of novel benzamides identified a panel of ~30 compounds with variable antimalarial activity against P. falciparum in vitro (IC50 0.048 – 6.48µM) (Wu et al., 2009). However, further studies are needed to elucidate the mechanism of action of these new compounds.

Chapter 1

1.11 Summary, hypotheses, and aims

There is significant interest in identifying new drug targets to address the current problem of malaria parasite drug resistance. HDAC enzymes, which are involved in transcriptional regulation, cell cycle regulation, cell differentiation, apoptosis and other essential processes in eukaryotic cells (Wade, 2001), are now under investigation as new antimalarial drug targets.

The P. falciparum parasite has at least five putative HDAC encoding genes, including three class I/II homologues and two class III homologues that are not essential to parasite survival in vitro (Duraisingh et al., 2005, Tonkin et al., 2009). The class I HDAC homologue in P. falciparum, PfHDAC1, is the focus of this thesis. Although PfHDAC1 is known to be expressed in asexual stages and localized to the parasite nucleus, little is known about the function or significance of this protein in malaria parasites.

The hypothesis of this study is that HDACs are important enzymes to P. falciparum survival and that inhibitors can be designed to specifically target these enzymes.

The specific aims of this project are:

1) To evaluate the antimalarial efficacy of new hydroxamate-based HDAC inhibitors against malaria parasites in vitro, in vivo and in combination with antimalarial drugs;

2) To investigate when and how hydroxamate-based HDAC inhibitors act against P. falciparum parasites; and

3) To characterize PfHDAC1 as a potential antimalarial drug target.

Chapter 2

General materials and methods

Chapter 2

2.1 P. falciparum in vitro cultures

P. falciparum lines used in this study are listed in Table S1. P. falciparum infected erythrocytes were cultured in O positive blood in RPMI 1640 (Gibco, USA) supplemented with 10% heat- inactivated pooled human sera, 50µg/mL hypoxanthine (Sigma, USA), and 80ng/mL gentamicin

0 (Pfizer, Australia). Cultures were maintained at 37 C in gas mixture composed of 5% O2, 5%

CO2, and 90% N2, as described previously (Trager & Jensen, 1976). Cultures were maintained between 0.1 – 10% parasitemia and 5% hematocrit, and monitored daily via microscopic examination of Giemsa-stained thin blood smears. Parasite cultures were fed by replacing the old culture media with freshly prepared complete media (as described above) every 1 – 2 days.

Alternatively, the parasite cultures were split by diluting a fraction of the blood pellet in new O positive blood and complete culture media to 5% hematocrit.

2.2 In vitro P. falciparum growth inhibition assays

Growth inhibition assays on in vitro cultured P. falciparum infected erythrocytes were carried out using an isotopic micro titre test, as described previously (Andrews et al., 2000). Briefly, test compounds were serially diluted, in triplicate wells, across wells of a 96-well micro titre plate in 100µL hypoxanthine-free culture media. Synchronous ring-stage P. falciparum cultures at 0.25% parasitemia – 2.5% hematocrit (100µL) were then added to each well. Red blood cells

(negative control) and parasite controls with and without compound vehicle (positive control) in triplicate wells were included on each assay plate. Plates were incubated for 48 h at 370C and

3 99% humidity in a gas mixture (5% O2, 5% CO2, 90% N2), then 0.5µCi of H-hypoxanthine

(Perkin-Elmer, USA) diluted in hypoxanthine-free culture media was added to each well. Plates were incubated for further 24 h prior to harvesting on 1450 MicroBeta filtermats (Wallac,

Finland). 3H-hypoxanthine incorporation was determined by rehydrating filtermat in BetaPlate scintillation solution (Perkin-Elmer, USA), sealed in plastic bags and measured in Beta liquid scintillation counter (Perkin-Elmer, USA). The final concentration of compound required to

3 inhibit 50% H-hypoxanthine incorporation (IC50) compared to control was determined by linear interpolation of inhibition curve as described previously (Huber & Koella, 1993).

Chapter 2

2.3 Blood and sera preparation

Human erythrocytes (O positive) obtained from the Australian Red Cross Services were washed three times in RPMI1640 media (Gibco, USA) by centrifugation at 726 rcf for 10 min. The washed blood was resuspended in complete culture media (Section 2.1) and stored at 40C for up to two weeks. Human plasmas obtained from the Australian Red Cross Services were pooled (5

– 10 units) and CaCl2 added to 2mM final concentration. Pooled plasma was transferred to

50mL conical tube and the mixture incubated at 370C until completely clotted. The clotted mixture was shaken vigorously, centrifuged at 3220 rcf for 15 min, and then heat-inactivated at

560C for 45min. The sera were stored at -200C in 50mL aliquots.

2.4 Cryopreservation of P. falciparum infected erythrocytes

Culutres containing predominantly ring-stage in P. falciparum infected erythrocytes were collected by centrifugation at 726 rcf for 2 min and culture media was removed. An equal volume of cryopreservation solution (0.65% NaCl, 3% Sorbitol, and 28% glycerol diluted in deionised water then autoclaved to sterilise) to packed red blood cell pellet was combined then cells transferred into a labeled 1.5mL cryovial tube (Greiner Bio-one, Belgium) and cells immediately frozen in an ethanol-dry ice bath for at least 15 min. The cryovials were then stored in liquid nitrogen.

2.5 Thawing P. falciparum infected erythrocytes

Cryovials containing cryo-frozen P. falciparum infected erythrocytes were removed from liquid nitrogen and rapidly thawed at 370C for 2 min. Cells were then transferred into a 50mL sterile conical tube (Greiner Bio-one, Belgium) and 0.2 pellet volume of 12% NaCl was added slowly drop-by-drop while continuously shaking the tube. Cells were incubated at room temperature for 5 min, then 10 volumes of 1.6% NaCl was slowly added drop-by-drop to the mixture. Cells were then pelleted by centrifugation at 726 rcf for 5 min, with no brake. The supernatant was removed and 10 volumes of 0.9% NaCl was slowly added drop-by-drop, the solution

Chapter 2 centrifuged as above and the supernatant removed. Cells were then washed once in 10 volumes of culture media, the returned to culture at 5% hematocrit (Section 2.1).

2.6 Sorbitol synchronization

Ring-stage P. falciparum infected erythrocytes were enriched by sorbitol treatment as described previously (Lambros & Vanderberg, 1979). Briefly, P. falciparum infected erythrocytes cultures were pelleted by centrifugation at 726 rcf for 2 min in a 10 – 50mL conical tube and medium was removed. The packed red blood cells pellet was resuspended in 10 volumes of 5% sorbitol solution (diluted in deionised water and autoclaved to sterilise) then incubated at room temperature for 5 min. The suspension was pelleted by centrifiguation and the sorbitol solution removed. The ring-stage enriched red blood cells pellet (>90% rings) was resuspended in complete culture media and returned to culture (Section 2.1).

2.7 Saponin lysis

P. falciparum infected erythrocyte cultures were pellet by centrifugation at 726 rcf for 2 min and the culture medium removed. Parasites were extracted by resuspending the culture pellet in

10 volumes of 0.15% saponin (Sigma, USA; diluted in PBS) and incubating on ice for 5 min.

The lysed red blood cell mixture was transferred into microfuge tubes (Eppendorf, Germany) and centrifuged at 16100 rcf at 40C for 5 min. The resulting pellets were washed three times in a large volume of cold-PBS to remove excess heam (or washing was continues until supernatant was clear). The parasite pellet was stored at-20 – -800C until needed.

2.8 Nuclear protein extracts

Nuclear protein extraction was carried out using a NucBuster protein extraction kit

(Calbiochem, USA) as per manufacturer’s instructions. Briefly, the parasite pellet (Section 2.7;

~1 × 109 parasites) was extracted in 100µL buffer 1 (provided with the kit) by vortexing for 15

– 30 sec, followed by incubation on ice for 5 min, and vortexing again. The sample was pelleted by centrifugation at 16100 rcf at 40C for 5 min. The soluble fraction containing the parasite

Chapter 2 cytosolic material was transferred to a fresh tube, and the pellet re-extracted in 50µL of buffer 2

(provided with the kit) as described above. The soluble fraction containing the parasite nuclear material was transferred to a fresh tube. All fractions and insoluble parasite pellets were stored at -800C.

2.9 Protein quantification

Protein concentrations were determined using the Bradford protein estimation method

(Bradford, 1976). Samples were diluted in a total volume of 50µL in 0.9% NaCl. After addition of 200µL Bradford solution (BioRad, USA) to each well in 96-well plate, the samples were incubated for 15 min at room temperature. Absorbance of samples was measured at 595nm in a

PolarStar plate reader (BMGLabtech, USA) in 96 well micro titre plates. Protein concentrations were calculated in comparison with a bovine serum albumin standard curve (0.5 – 10µg BSA, in triplicate measurements).

2.10 Deacetylase activity assays

Parasite nuclear extracts (Section 2.8; ~1 × 108 parasites) were used in each reaction in a fluorometric deacetylase activity assay kit (Upstate, USA). Each assay was carried out in duplicate reactions with each reaction containing 15µL substrate (provided with the kit), 10µL assay buffer or inhibitor, and 15µL nuclear extracts in final volume of 40µL. In some cases, inhibitors were included in the reaction. Deacetylase reactions were carried out at 370C, in the dark, for 1 h. A 20µL volume of developer reagent (provided with the kit) was then added to each well to stop the reaction. Deacetylase activity was measured as arbitrary fluorescence units

(AFU) at 370nm (emission) and 460nm (excitation) in a PolarStar plate reader (BMG Labtech,

USA).

2.11 Western blot

P. falciparum parasite pellet (Section 2.7) was resuspended in sodium dodecyl-sulfate polyacrylamide gel electrophoresis sample buffer (1× SDS-PAGE buffer), heated at 900C for 3

Chapter 2 min, then briefly centrifuge for 1 min. Protein extract was separated via 10 – 12% SDS-PAGE and then protein transferred to polyvinylidene fluoride (PVDF) membrane (Roche, Germany).

The membrane was pre-blocked in BLOTTO (3 – 5% skimmilk-PBS solution) for at least 1 h at room temperature and then incubated in BLOTTO containing primary antibody (diluted to

1:1000 – 1:10000) for at least 1 h at room temperature or overnight at 40C on a tube roller.

Three 10 min washes with 1×PBS-Tween 20 (0.05%) were carried out prior to incubation in secondary antibody conjugated to horseradish peroxidase (diluted to 1:5000 in BLOTTO,

Zymed, USA) for 45 min at room temperature. The membrane was washed as described above and then incubated in enhanced chemiluminescence solution (ECL; Amersham, UK) for 5 min.

Excess ECL was drained, the membrane was sandwiched between two plastic sheets and the membrane exposed to X-ray film (FujiFilm, Japan) and processed in a Kodak 3000RA XOMAT developer.

2.12 Stripping PVDF (Western blot) membranes

Membrane was stripped in stripping buffer (62.5mM Tris, pH6.8, 2% SDS, 100mM β- mercaptoethanol) at 500C with constant rotation in a glass bottle for 30 min and then washed three times in 1×PBS. The stripped membrane was incubated in ECL, drained and sandwiched between plastic sheets. To ensure that stripping was successful the membrane was exposed to

X-ray film and processed as above (Section 2.11). Membrane was blocked in BLOTTO before re-using for Western blot analysis.

2.13 RNA extraction

P. falciparum parasite pellet (Section 2.7; ~1 × 108 parasites) was resuspended in 1mL TRIzol solution (Invitrogen, USA), then 0.2 volume of chloroform was added and the solution mixed thoroughly by brief vortexing. The sample was then centrifuged at 16100 rcf at 40C for 30 min.

Following centrifugation the upper layer containing the RNA was carefully aspirated and transferred to a microfuge tube (Rnase- and Dnase-free) and the RNA precipitated by adding 1.5 volumes of 100% isopropanol. Following centrifugation, as described above, the supernatant

Chapter 2 was decanted and the RNA pellet was washed in 500µL of 70% ethanol. The RNA pellet was allowed to briefly air-dry at room temperature and then resuspended in 30 – 50µL of formamide and stored at -800C.

2.14 Northern blot

Northern blots were carried out as previously described (Kyes et al., 2000). Briefly, 5 – 10µg of

RNA was separated via 0.8% agarose gel electrophoresis in 1×TBE buffer (0.089M Tris,

0.089M Boric acid, 2mM EDTA, 5mM guanidine thiocyanate) at 110V for 15 min then 80V for

1 – 2 h. The gel was stained with 0.5µg/mL ethidium bromide for 5 min and a photo taken, alongside a ruler to record ribosomal band intensity for equal loading in each lane. The gel was then soaked in 7.5mM NaOH twice for 10 min and RNA were transferred to nitrocellulose membrane (N-hybond, Amersham, UK) by the capillary method in 7.5mM NaOH overnight.

The membrane was neutralised in 2×SSC buffer (0.3M NaCl, 0.03M sodium citrate) for 5 min and allowed to air dry prior to UV-crosslinking RNA onto membrane at 125mJoules in UV- crosslinker (Stratagene, USA). The membrane was prehybrised for 3 h in pre-hybridisation buffer (7% SDS, 0.5M sodium phosphate (NaH2PO4, pH7.2), 2% Dextran sulfate) then hybridised at 420C overnight in same solution containing 32P-dATP labeled probes (Fermentas,

USA). 32P-dATP labeling of probes was carried out according to the manufacturer’s instructions. The membrane was then washed three times at 420C in wash buffer (0.5×SSC,

0.1% SDS), wrapped in plastic wrap to keep moist, and exposed to X-ray film (FujiFilm, Japan) with an intensifying screen at -800C for 1 – 48 h.

2.15 Genomic DNA extraction

P. falciparum parasite pellet (Section 2.7) was washed once in 1mL of TKM1 buffer (10mM

Tris, pH7.6, 10mM KCl, 10mM MgCl2, and 2mM EDTA) for every 50µL packed cell pellet.

The pellet was resuspended in 100µL of TKM1 buffer and then 400µL of TKM2 buffer (10mM

Tris, pH7.6, 10mM KCl, 10mM MgCl2, 2mM EDTA, and 0.4M NaCl) was added. The sample was mixed by vortexing, then 25µL of 20% SDS added, followed by incubation at 560C for 15

Chapter 2 min. Next, 150µL of 6M NaCl was added, the sample centrifuged for 5 min at 16100 rcf and the soluble fraction containing the genomic DNA transferred to a microfuge tube. To precipitate

DNA, 2 volumes of 100% ethanol was added and the sample centrifuged at 40C for 15 min. The supernatant was then decanted and the DNA pellet washed once in 500µL of 70% ethanol, air dried, and then resuspended in 50µL of sterilised deionised water or TE buffer (10mM Tris, pH8.0, and 1mM EDTA). DNA was stored at -200C.

2.16 Polymerase chain reaction (PCR)

All PCR reactions were carried out in 25 – 50µL reaction containing 1× PCR reaction buffer, 1

– 2mM MgCl2, 200µM dNTPs (Peqlab, Germany), 200nM primers (forward and reverse), 2.5 units Taq polymerase (AmpliTaq GOLD, Applied Biosystem, USA) or Pfu Taq polymerase

(Strategene, USA) and 20 – 50 ng DNA template. For PCR cycling, samples were first denatured at 950C for 5 min, then 30 cycles of denaturation (950 for 30 sec), annealing (45 –

550C for 30 sec) , and DNA extension (680C) for 3 – 4 min, a final extension step was carried out at 680C for 10 min then the samples were stored at 40C.

2.17 DNA Sequencing

Sequencing was carried out using ABI Bigdye version 3.1 (Applied Precision, Australia) in quarter reaction volume. A reaction mix consisted of 1.2µL of dye terminator ready reaction mix, 3.2pmol of primer, 20ng of template, and milliQ water to a final volume of 12µL per reaction. The sample was subjected to 25 cycles of denaturation (960C for 30 sec), annealing

(500C for 15 sec), and extension at 600C for 4 min. Isopropanol precipitation was carried out following the sequencing reaction to remove dye terminator. The sequencing reaction was extracted with 120µL of 70% isopropanol, incubated at room temperature for 10 min, then centrifuged at 16100 rcf for 30 min in a bench top centrifuge (Eppendorf, USA). The supernatant was discarded by aspiration, and the pellet washed with 500µL 70% isopropanol followed by centrifugation at 13200rpm for 30 min. The supernatant was removed by gentle aspiration and the pellet dried in a SpeedyVac vaccumn for 15 min at 300C. DNA sequence

Chapter 2 analysis was carried out at the QIMR DNA and peptide purification unit using the ABI capillary system.

2.18 pGEM-T ligation

The pGEM-T ligation system (Promega, USA) was used to ligate PCR product to the pGEM cloning vector. For PCR product amplified with Pfu Taq polymerase (PCR clean up using the

Qiagen PCR Clean-up kit, as per manufacturer’s instructions), an “A” nucleotide was added to the 3’ primed end of the blunt product by incubating with Taq DNA polymerase at 700C for 30 min in 1× Taq buffer, 1 – 2mM MgCl2, and 200nM dATP. A molar ratio of insert:vector of 3:1 was used in the ligation mix in accordance with the kit recommendation. Ligation was carried out as per manufacturer’s instructions (pGEM-T cloning kit, Promega, USA).

2.19 Plasmid extraction

E. coli were grown overnight at 370C in Luria Broth (LB) containing the appropriate antibiotic

(up to 2mL culture volume) and pellet by centrifugation at 13200rpm for 10 min in a bench top centrifuge (Eppendorf, USA). Mini-prep plasmid extractions (5 – 10µg) were carried out using the Roche High Pure Plasmid Isolation kit (Roche, Germany) as per manufacturer’s instructions. Plasmid DNA was eluted in 30 – 50µL EB buffer (provided with the kit) or sterilised deionised water and stored at -200C. For midi-prep plasmid extraction, E. coli cells were grown in LB as described above (50 – 100mL culture volume) and cells were collected by centrifugation at 16100 rcf for 10 min in a high speed centrifuge (Bechman and Coulter, USA).

Plasmid DNA was isolated from cells using the Midi-prep plasmid kit (Qiagen, USA) as per manufacturer’s instructions. Midi-preparations of plasmid DNA were resuspended in 500µL sterilised deionised water and store at -200C.

2.20 Restriction digestion

Restriction digest of DNA was carried out in a 50µL reaction volume containing 0.5 – 1µg

DNA, 10 – 20 units of restriction enzymes and 1× reaction buffer (provided with enzymes, New

Chapter 2

England Biolabs, UK). In some cases, depending on the enzyme, BSA was included in the reaction mix to stabilize the enzyme. Restriction digest reaction was carried out at 370C for 2 h or overnight.

2.21 DNA gel electrophoresis

DNA (0.5 – 1µg) was separated on 0.8 – 1% agarose gel at 80 – 100V in 1× TAE buffer containing 0.5µg/mL ethidium bromide. Agarose gel was analysed using the Gene Genius Bio

Imaging System and images were acquired using the Genesnap software (Syngene, USA).

2.22 Gel purification

Extraction o DNA bands from agarose gel were carried in a microfuge tube using the Qiagen

Gel Purification kit as per manufacturer’s instructions (Qiagen, USA). DNA was eluted out in

30 – 50µL EB buffer (provided with the kit) and stored at -200C or until use.

2.23 E. coli transformation

Plasmid DNA was transformed into TOP10 E. coli cells (Invitrogen, USA) by chemical transformation of 0.1 – 0.5µg of plasmid. DNA was added to 1 – 2 × 109 competent cells in a

100µL volume and the samples mixed gently by stirring with a pipette tip. The mixture was immediately incubated on ice for 10 min, then heat-shocked at 420C for 30 – 60 sec for uptake of the plasmid. The tube was then immediately transferred to ice for 2 min, then 900µL of SOC media (2% tryptone, 0.5% yeast extract, 10mM NaCl, 2.5mM KCl, 2% glucose) added and the transformation mixture incubated at 370C for 1 h, with shaking (200rpm in a Bioline Shaker,

Edwards Instrument Company Australia, Australia). A sample of the transformation mixture

(100 – 200µL) was then plated onto LB agar plates containing an appropriate antibiotic and the plates incubated at 370C overnight to obtain single colony.

Chapter 2

2.24 P. falciparum transfection

Transfection was carried out by electroporation of synchronous ring-stage P. falciparum infected erythrocytes essentially as previously described (Tonkin et al., 2004). A 10% parasitemia culture of P. falciparum in 200µL volume was combined with 700µL of cytomix

(120mM KCl, 0.15mM CaCl2, 2mM EGTA, 5mM MgCl2, 10mM K2HPO4, 25mM HEPES pH7.6) containing 50 – 100µg of plasmid DNA (in 30 - 50µL TE buffer, 10mM Tris, pH8.0,

1mM EDTA) then transferred to a 0.2cm2 electrocuvette (Biorad, USA). The mixture was then electroporated using the following conditions: 2.5kV, 200Ω, and 2.5µF in a cell electroporator

(Biorad, USA). Following electroporation the mixture was then returned to culture in freshly prepared 10mL parasite media containing 300µL of O positive human erythrocytes. The culture was grown for 48 h prior to addition of 5nM of WR99210. The culture was maintained by replacing fresh culture media containing 5nM of WR99210 daily, up to day 10 post transfection.

Thereafter, the culture was maintained on 5nM of WR99210 with two media changes on a weekly basis. Positively selected parasites were observed in cultures between days 20 – 30 post transfection via light microscopic analysis of Giemsa-stained thin blood smears. Stocks of parasite cultures were grown and cryopreserved as described in Section 2.4.

2.25 Immunofluorescent assays (IFA)

P. falciparum infected erythrocyte cultures were harvested by centrifugation at 726 rcf for 2 min. The pellet was washed three times in 1× PBS (Gibco, USA) to remove the culture medium, then diluted to 60 – 70% hematocrit and thin blood smears were prepared on glass slides and then dried. The smears were fixed in cold 10% methanol-acetone for 30 min and then dried.

Slides were marked with a Pap-Pen (SCI Science Services, Germany) which formed wells. Cells were blocked overnight in 3% bovine serum albumin (BSA) solution (diluted in PBS) at 40C.

Cells were then incubated with 1:500 dilution of primary antisera diluted in 3% BSA solution at room temperature for 45 min. Slides were washed three times with PBS, then anti-mouse IgG conjugated to Cy2 antisera (diluted to 1:500, Sigma, USA) in 3% BSA solution added to each well. Following incubation for 45 min at room temperature in the dark, slide were washed as

Chapter 2 above. To label nucleic acid, 0.5µg/mL Hoechst diluted in 3% BSA solution was added to each well and slides incubated for 10 min in the dark. Slides were washed again, as above, and wells were mounted in 70% glycerol under a coverslip. Images were captured using an AxioSkop 2

Mot Plus microscope (Ziess, USA).

Chapter 3

In vitro and In vivo antimalarial activity of L-cysteine and 2-

aminosuberic acid HDAC inhibitor analogues

Publication arising from this work:

Andrews KT, Tran TN*, Lucke AJ, Kahnberg P, Le GT, Boyle GM, Gardiner DL, Skinner-

Adams TS, Fairlie DP: Potent antimalarial activity of histone deacetylase inhibitor analogues.

Antimicrobial Agents and Chemotherapy 2008, 52(4):1454-1461.

*Co-first Author: Equal contribution to manuscript

Chapter 3

3.1 Introduction

The most effective treatment currently available for uncomplicated malaria caused by P. falciparum is artemisinin-based combination therapy (ACT) which combines artemisinin (or one of its derivatives) with a second antimalarial drug with a longer half life. Recent reports show a declining efficacy of ACT on the Thai-Cambodia border (Wongsrichanalai & Meshnick,

2008, Noedl et al., 2008, Dondorp et al., 2009), an observation that underscores the need to develop alternative combinations of existing antimalarial drugs and new agents that act on novel targets within the parasite. P. falciparum HDACs are one such new antimalarial target.

PfHDACs were first identified as a new drug target in malaria parasites when it was found that inhibitors of mammalian histone deacetylase (HDAC) enzymes including trichostatin A (TSA) and apicidin kill P. falciparum parasites (Darkin-Rattray et al., 1996). Apicidin was shown to be orally active against rodent malaria parasites (Darkin-Rattray et al., 1996) and appears to target

PfHDAC activity as the compound was shown to cause hyperacetylation of parasite histones.

Unfortunately TSA and apicidin do not selectively kill malaria parasites as they also inhibit mammalian cell growth at similar concentrations (Darkin-Rattray et al., 1996, Glenn et al.,

2004). Therefore, if HDAC inhibitors are to be useful antimalarial agents, next generation compounds need to demonstrate better parasite selectivity. SAHA and other synthetic hydroxamate-based HDAC inhibitors including azelaic bishydroxamic acid (ABHA) and suberyl bishydroxamic acid (SBHA) have also been shown to be active against P. falciparum in vitro (Andrews et al., 2000, Mai et al., 2004) with ABHA and SBHA being more parasite selective than apicidin and TSA (Table 1.4). Intraperitoneal administration of SBHA in a mouse malaria model was able to delay and reduce parasitemia (Andrews et al., 2000).

Recently, a panel of new synthetic non-peptidic hydroxamate-based compounds derived from L- cysteine and 2-aminosuberic acid have been investigated as antitumour agents and have been shown to have selective and potent activities against cancer cells (IC50 range 10 – 600nM) and up to 87 times more selectivity in killing cancer cells over normal cells (Glenn et al., 2004,

Kahnberg et al., 2006). In this study the antimalarial activity of these compounds was examined

Chapter 3 alone, and in combination with three antimalarial drugs in vitro against P. falciparum, and as a monotherapy in vivo in a mouse malaria model.

3.2 Materials and methods

3.2.1 Compounds

The synthesis and characterisation of the L-cysteine and 2-aminosuberic acid hydroxamate derivatives have been reported elsewhere in connection with their antitumour activities

(Kahnberg et al., 2006, Glenn et al., 2004). These compounds contain two side chain groups (R1 and R2) and are substituted at the X position (Figure 3.1) with either a thiolether sulfur group (X

= S) or a methylene group (X = CH2) group to produce L-cysteine (L-CYS) or 2-aminosuberic acid (2-ASA) compounds, respectively. All inhibitors contain a hydroxamic acid group to ensure tight zinc metal ion binding in the active site of the targeted HDAC enzymes (Figure

3.1). These compounds were provided by Prof. David Fairlie (IMB). All compounds were prepared as 5 – 20mM stocks in 100% dimethyl sulfoxide (DMSO) and stored at -200C.

O O H H R1 N R2 R1 N R2 N N H H O O S CH X 2 X

O O ZBG ZBG HN HN OH OH L-cysteine 2-aminosuberic acid

Figure 3.1 Schematic representation of non-peptidic compounds derived from L-cysteine and 2-aminosuberic acid. Shown are capping groups, R1, acid group side chain, and R2, amine group side chain and a hydroxamic acid zinc binding group (ZBG, arrows) separated by hydrophobic linker region. X = S (thiolether sulfur) or X = CH2 (methylene).

3.2.2 P. falciparum in vitro growth inhibition assays

P. falciparum Dd2 (Wellems et al., 1988), 3D7 (Walliker et al., 1987), PH1 (Chen et al., 2005) and K1 (Burkot et al., 1984) were cultured in vitro with O positive human erythrocytes, as described (Section 2.1). Growth inhibition assays of synchronous ring-stage infected erythrocytes starting at 2.5% hematocrit and 0.25% parasitemia was assessed using 3H- 38

Chapter 3 hypoxanthine incorporation, as described (Section 2.2). Chloroquine was included in all assays as an internal control. The final concentration of each compound required to inhibit 3H- hypoxanthine incorporation by 50% compared to vehicle controls (IC50), was determined by log-linear interpolation of inhibition curves (Huber & Koella, 1993). Each compound was assayed in triplicate, on at least three separate occasions. Data are presented as IC50 (± standard deviation, SD). To calculate the statistical significance between strains, the Kruskal-Wallis test with Dunn’s Multiple Comparison test was carried out.

[Note: assays using PH1 and K1 were carried out by Dr Kathy Andrews (QIMR) and are included for comparison.]

3.2.3 Mammalian cell toxicity assays

The effect of HDAC inhibitors on neonatal foreskin fibroblast cells (NFF) and human lung endothelial cells (HLEC) was determined using a clonogenic cell survival assay and carried out by Dr Glen Boyle (QIMR) in collaboration.

3.2.4 Oral pharmacokinetic studies in mice

The oral bioavailability of 2-ASA-14 was examined in BALB/c mice. Six groups of two mice were each administered 5mg/Kg of 2-ASA-14 dissolved in 50% DMSO in a 50µL volume by oral gavage. Mice were euthanized by CO2 at 0, 20, 40, 60, 120, and 240 minutes following administration of compound and blood collected by heart puncture. Plasma was prepared and spiked with an analogue of 2-ASA-9 (0.5µg/mL) as internal standard, then sera aliquoted, and stored at -800C. The inclusion of the internal standard (with a different molecular weight) was to ensure that compound stability was not affected by storage and to compensate for any loss of compound during preparation of plasma samples for analysis. A standard curve was also prepared by diluting different concentrations of 2-ASA-14 (0.5, 1, 2, 5, 10, and 20µg/mL) in mouse sera. Each standard sample was also spiked with 0.5µg/mL of 2-ASA-9 internal standard. Samples were prepared for liquid chromatography-tandem mass spectrometry (LC-

MSMS) by combining 50µL of plasma each sample with 50µL of internal standard (2-ASA-9 at

Chapter 3

0.5µg/mL) and then extraction with HPLC grade dichloromethane, evaporation and reconstitution in mobile phase (a carrier for the sample solution). Samples were analysed on a

C18 Phenomenex Luna column (50x2.1 mm) using acetonitrile/water gradients and detected by electrospray mass spectrometry (Sciex API-3000) as previously described (Deng et al., 2009).

Compound concentrations were determined using the internal standard ratio from the standard curve. Mass spectrometry analysis of prepared sera samples was carried out in collaboration with Dr Bob Reid (Institute for Molecular Bioscience, UQ). Animal work was carried out at the

QIMR Animal Facility under AEC approval number P752 (A0404-065M).

3.2.5 In vivo antimalarial studies in mice

The antimalarial efficacy of 2-ASA-9 and 2-ASA-13 was examined in BALB/c mice infected with P. berghei malaria parasites, which produces a lethal infection (Carter & Diggs, 1977), as described previously (Andrews et al., 2000). Groups of six mice were infected with 106 P. berghei infected erythrocytes, passage from an infected mouse. HDAC inhibitors (prepared in

50% DMSO at 10mg/kg in 100µL volumes) were administered by oral gavage, twice daily for three days, beginning 2 h post infection (p.i.). An antimalarial control group received 10mg/kg chloroquine dissolved in 100µL 50% DMSO by oral gavage, once daily for three days beginning 2 h p.i. A vehicle control group received 100µL of 50% DMSO in PBS by oral gavage, twice daily for three days beginning 2 h p.i. Peripheral parasitemia was monitored daily, beginning 3 days p.i., via Giemsa-stained thin blood smears prepared from tail snips.

Parasitemias for the 6 mice in each group, at each time point, (± standard deviation of counts by two independent persons) were determined. Statistical significance was determined by non- parametric Mann-Whitney test (P≤0.05). Animal work was carried out at the QIMR Animal

Facility under AEC approval number P752 (A0404-065M).

3.2.6 Isobologram assays

In vitro drug interactions were assessed using isobologram analysis as previously described

(Skinner-Adams et al., 2007). Briefly, the IC50 values for each compound were determined and

Chapter 3 used as the fractional inhibitory concentration (FIC = 1.0). FICs were then titrated against a range of each of the compounds to be tested. All assays were performed in 96-well micro titre plates with each well containing 100µL of culture (ring-stage Dd2 infected erythrocytes at

0.25% parasitemia and 2.5% hematocrit) and 100µL of a compound dilution or control and cultured under standard conditions for 48 h prior to addition of 3H-hypoxanthine (0.5µCi to each well; Amersham, GE healthcare, UK). Culture plates were grown for another 24 h prior to harvest onto MicroBeta1450 filtermatA filter paper (Wallac, Perkin-Elmer, USA) and counted in a 1450 MicroBeta Liquid scintillation and chemiluminescence counter (Perkin-Elmer, USA).

Experiments were repeated on at least two separate occasions. Isobolograms (plots of drug combinations with resulted in 50% growth inhibition) were constructed using data from each experiment (Berenbaum, 1978). Isoboles were constructed from these FIC values. Using the

SAAMII program (SAAM Institute, Seattle, WA, USA), and the standard hyperpolic function

(-I) Yi = 1 – [XI/(XI + e × (1-Xi))]

Where Yi the IC50 of drug A when combined with drug B, Xi the IC50 of drug B when combined with drug A. I, the interaction value was fitted to the data (Canfield et al., 1995). For additive interactions I = 1.0, for synergistic interactions I > 1.0 and for antagonistic interactions I < 1.0.

The significance of the difference of I from zero was assessed using student’s t-test.

3.3 Results

3.3.1 In vitro antimalarial activity of new hydroxamate-based HDAC inhibitor

compounds based on L-CYS and 2-ASA structures

A panel of fourteen compounds (six derived from L-CYS and eight derived from 2-ASA) were initially screened against a chloroquine resistant P. falciparum clone (Dd2). All six compounds from the L-CYS series inhibited P. falciparum (Dd2) growth with IC50 values between 48 –

399nM (Table 3.1). Three of the six compounds display ≥7 times more selectivity in killing parasites over a normal cell line (NFF). The eight compounds from the 2-ASA series were

Chapter 3 generally more potent than the L-CYS inhibitors with IC50 values against Dd2 of 33 – 105nM

(Table 3.2). These compounds were also more selective than those compounds from L-CYS series (Selectivity range 4 – 167). Six compounds from the 2-ASA series demonstrated IC50 values <100nM against Dd2. The antimalarial activity of these 2-ASA analogues were also tested against three additional P. falciparum strains, a chloroquine sensitive (3D7, this study) and two chloroquine resistant (PH1 and K1, Dr. Kathy Andrews, personal communication) strains. For all compounds Dd2 tends to have higher IC50 values compared to the three other lines. However, due to low number of assay replicates this difference is only significant for 2-

ASA-7 and 2-ASA-9 (p <0.04) and approaching significance for 2-ASA-12 and 2-ASA-14 (p

<0.075) (Table 3.2). The inhibitory activity of these compounds was also tested against an additional mammalian cell line (human lung endothelial cells, HLEC). All eight compounds were less potent against HLEC cells with exception to compound 2-ASA-10 which was 3 times more selectivity for NFF over HLEC (Table 3.2). Compounds 2-ASA-9, 2-ASA-11, 2ASA-12, and 2-ASA-13 were >100 times more active in killing parasites than mammalian cells (Table

3.2).

Chapter 3

Table 3.1. Comparative toxicities of L-CYS deratives against P. falciparum and mammalian cells. P. falciparum IC of Compound a R1 a R2 LogD Dd2 50 SIc 7.0 NFF nMb IC50 nM (SD)

L-CYS-1 Br 2.6 125 (35) 830 13

L-CYS-2 2.4 339 (219) 800 2

L-CYS-3 N 1.8 169 (149) 350 2

L-CYS-4 N N 2.0 48 (43) 600 12

N L-CYS-5 2.8 305 (209) 2200 7

N 3 L-CYS-6 3.7 320 (50) 320

a R1, acid group side chain, and R2, amine group side chain on L-cysteine core structure as shown Figure 3.1. b Mammalian cells toxicities against neonatal foreskin fibroblast cells were previous reported in (Glenn et al., 2004) and included for comparison. SD: standard deviations c Selectivity (selectivity index; SI) calculated as the difference between P. falciparum and mammalian cell toxicity IC50 values (IC50 of mammalian cells/IC50 of P. falciparum).

Chapter 3 Table 3.2. Comparative toxicities of 2-ASA derivatives against P. falciparum versus mammalian cells. a 1 a 2 Compound R R LogD7.0 P. falciparum Mammalian cells Selectivity b c d IC50 nM (SD) IC50 nM range Dd2 3D7 PH1 K1 NFF HLEC

2-ASA-7 4.8 105 (47) 29 (22) 56 (4) 47 (14) 2200 2553 21-88 O

2-ASA-8 Br 5.3 41 (22) 22 (14) 33 (7) 30 (3) 190 2043 5-93

2-ASA-9 3.2 39 (24) 15 (9) 25 (1) 20 (5) 1240 1954 32-130

Br 2-ASA-10 3.0 34 (164) 91 (97) >500 >500 5860 1889 4-64

2-ASA-11 2.7 102 (44) 34 (17) 48 (3) 54 (31) 4010 5667 39-167

2-ASA-12 2.3 63 (42) 34 (17) 44 (8) 40 (0) 1260 3551 20-104 N H

2-ASA-13 N 2.8 71 (65) 19 (10) 31 (1) 25 (12) 570 2513 8-132 N

2-ASA-14 2.9 33 (33) 13 (12) 27 (6) 19 (2) 337 950 10-50 N H N aR1, acid group side chain, and R2, amine group side chain on 2-aminosuberic acid core structure as shown Figure 3.1. bP. falciparum toxicities against PH1 and K1 clones were carried out by Dr Kathy Andrews, QIMR (unpublished data) and included for comparison. SD: standard deviations cMammalian cells toxicities against neonatal foreskin fibroblast cells were previous reported in (Kahnberg et al., 2006); against human lung endothelial cells (HLEC) carried out by Dr Glen Boyle, QIMR (unpublished data), and included for comparison. d Selectivity calculated as the difference between P. falciparum and mammalian cell toxicity IC50 values (IC50 of mammalian cells/IC50 of P. falciparum). 44

Chapter 3 3.3.2 Oral bioavailability of 2-ASA-14

Oral bioavailability studies were carried out in mice using compound 2-ASA-14. This compound was selected as a representative of 2-ASA compound as mg quantities were available for testing. 2-ASA-14 has a LogD7.0 of 2.9, which is the octanol/water partition coefficient as calculated using PALLAS (Glenn et al., 2004). LogD7.0 values between 1 – 5 indicate that suitable penetration of cell membrane is likely to be achieved. When given as a single dose of

5mg/kg, the maximum concentration (Cmax) of 2-ASA-14 reached was 340ng/mL (~720nM; n=2), ~20 times the in vitro IC50 value against P. falciparum (Figure 3.2). This maximum concentration was reached within 1 h after administration (Tmax) and was rapidly cleared within

2 h.

400

300

Concentration

200

(ng/mL) Plasma 100

Mean

0 0 204060120240

Time (min)

Figure 3.2. Oral bioavailability of 2-ASA-14 in BALB/c mice. Mean plasma concentrations (n=2) of compound over time are shown as determined by LC-MSMS. The Cmax and Tmax were determined to be 340ng/mL (720nM) and 60 min, respectively.

3.3.3 In vivo antimalarial efficacy of 2-ASA compounds in a mouse malaria

model

The in vivo antimalarial activity of compounds 2-ASA-9 and 2-ASA-13 were tested in a mouse malaria model. These compounds were selected as mg quantities needed for these studies were available. Compounds were administered to groups of six mice, orally by gavage beginning 2 h post infection (p.i.) with 106 P. berghei infected erythrocytes. Dosing was twice daily for three days (10mg/kg/dose). Compound 2-ASA-9 administration resulted in a small but statistically significant, reduction in peripheral parasitemia compared to untreated controls on day 6 and 7 p.i. (Figure 3.3, P<0.05, asterisk). The parasitemias in mice treated with 2-ASA-13 were not 45

Chapter 3 significantly different to that of control mice (P>0.05). The mean peak peripheral parasitemia of the control group was ~25% and was achieved after 7 – 8 days p.i. As expected, mice treated with a single 10mg/kg oral dose of chloroquine, once daily for three days, did not developed peripheral parasitemia (not shown). Mice did not develop any physical signs of toxicity (i.e. ruffled coat, hunched back) when treated with concentration of 10mg/kg/dose of compounds 2-

ASA-9 or 2-ASA-14. Furthermore, in another study mice treated with a hydroxamate HDAC inhibitor (WR301801) at 50mg/kg/day also demonstrated no observable signs of toxicity

(Agbor-Enoh et al., 2009).

Control 2-ASA-13 Control 2-ASA-9 35 35 30 30 * 25 25 20 20 15 15 10 10

% Parasitemia 5 % Parasitemia 5 0 0 012345678 012345678 Days Post Infection Days Post Infection

Figure 3.3 In vivo antimalarial efficacy of 2-ASA-9 and 2-ASA-13 in mice. Groups of six BALB/c mice were infected with 106 P. berghei infected erythrocytes then orally treated with 10mg/kg of 2-ASA9 or 2-ASA-13, twice daily for three days, beginning 2 h post infection. Vehicle control mice received 50% DMSO in PBS. Peripheral parasitemia was determined daily by microscopic examination of Giemsa-stained thin blood smears. The mean parasitemia of each group of mice are shown. Error bars indicate standard deviation. Each smear was counted by two different persons. A significant difference in parasitemia compared to control was observed on days 6 and 7 (p.i.) for 2-ASA-9 (P≤0.05, asterisk).

3.3.4 In vitro interactions of HDAC inhibitors and known antimalarial drugs

The in vitro interactions between the HDAC inhibitors SAHA or 2-ASA-9 with chloroquine, quinine, and artemisinin was examined using isobologram analysis (Section 3.2.6). Data from these studies demonstrated that interaction between SAHA and 2-ASA-9 is antagonistic (I = -

1.42, P < 0.0001; Figure 3.4A). Combinations of 2-ASA-9 with chloroquine (I = -4.44, P <

0.0001) or artemisinin (I = -0.92, P < 0.005) were also antagonistic (Figure 3.4). However, interactions between quinine and SAHA (I= -1.3, P = 0.1) or 2-ASA-9 (I = -1.52, P = 0.1) were weakly antagonistic (Figure 3.4).

Chapter 3

A SAHA and 2-ASA-9 B Chloroquine and 2-ASA-9 I = -1.42, P < 0.0001 I = -4.44, P < 0.0001 1.2 1.2 1 1 0.8 0.8 0.6 0.6 0.4 0.4 2-ASA-9 FIC 2-ASA-9 FIC 0.2 0.2 0 0 0 0.2 0.4 0.6 0.8 1 1.2 0 0.2 0.4 0.6 0.8 1 1.2 SAHA FIC Chloroquine FIC

C Quinine and 2-ASA-9 D Quinine and SAHA I = -1.52, P = 0.1 I = -1.31, P = 0.1 1.2 1.4 1 1.2 1.0 0.8 0.8 0.6 0.6

0.4 FIC SAHA 0.4 2-ASA-9 FIC 0.2 0.2 0 0.0 0 0.2 0.4 0.6 0.8 1 1.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 Quinine FIC Quinine FIC

E Artermisinin and 2-ASA-9 F I = -0.92, P < 0.005 1.2 1 0.8 0.6 0.4 2-ASA-9 FIC 0.2 0 0 0.2 0.4 0.6 0.8 1 1.2 Artermisinin FIC

Figure 3.4 In vitro antimalarial activity of HDAC inhibitors when combined with known antimalarial drugs. Combinations are SAHA and 2-ASA-9 (A), and choroquine and 2-ASA-9 (B), quinine and 2-ASA-9 (C), quinine and SAHA (D), or artemisinin and 2-ASA-9 (E) For additive interactions I =1.0, for synergistic interactions I>1.0, and for antagonistic interactions I<1.0 (F). P values represent significance of interaction (if I crosses the line additivity). All data points are shown for two independent assays for each drug combination.

Chapter 3 3.4 Discussion

Antimalarial drug resistance and the lack of a licensed malaria vaccine underscore the need to develop new drugs and/or drug combinations to limit the spread of drug resistance. P. falciparum HDACs were first recognised as potential antimalarial targets based on the observation that inhibitors of mammalian HDACs (apicidin and TSA) kill P. falciparum infected erythrocytes and cause hyperacetylation of parasite histones (Darkin-Rattray et al.,

1996). In this study, a panel of fourteen compounds, six derived from L-CYS and eight derived from 2-ASA, were assessed in vitro against P. falciparum and all were found to be active at low nM concentrations (IC50 13 – 339nM). The 2-ASA compounds were active against chloroquine sensitive (3D7) and chloroquine resistant (Dd2, PH1 and K1) P. falciparum lines. This observation suggests 2-ASA compounds might be active against drug resistant parasites in the field. While further experimental testing is required to ensure this is the case, these data bode well for the potential clinical use of HDAC inhibitors. The 2-ASA compounds are more potent than L-CYS compounds (up to 10 fold difference in activity), and this difference may be due to a combination of factors including the binding specificity of the capping groups, the length of hydrophobic linker region, and accessibility of drug to target. Previous studies have suggested that the S-C bond at X position of the core of structure (Figure 3.1) of the L-CYS compounds results in a linker length of five to six methelyne units which may provide too much conformational freedom to the enzyme-bound inhibitor at the rim of the tubular active site

(Kahnberg et al., 2006). Likewise, in another study (Patil et al., 2009) hydroxamate compounds containing ≥7 methylene units showed reduced antimalarial activity (>10 fold less activity than compounds with 5 – 6 methylene units) indicating that optimal linker length might be critical for tight interaction between enzyme-bound inhibitor in the active site of target HDAC enzymes. The linker region of 2-ASA compounds is five methylene units which more closely mimics the lysine side-chain of protein substrate (Kahnberg et al., 2006) and this may explain the improved antimalarial activity of this series compared to the L-CYS compounds. There are also other chemical and biological advantages for suberoyl compounds over L-CYS derived thioether group (X = S, Figure 3.1). The thioether sulfur is known to be a pi-acceptor that can

Chapter 3 coordinate to metal ions thereby potentially directing the compounds to other proteins or enzymes containing zinc or other metal binding groups (Kahnberg et al., 2006). These non- specific interactions with other proteins are likely to result in more biological side effects.

Furthermore, the thioether sulfur of L-CYS can be oxidised to produce sulfoxide, then sulfone which can act in free radical chemistry and may affect other downstream activity of other proteins (Kahnberg et al., 2006).

Given the potent in vitro antimalarial activity and promising parasite selectivity of the 2-ASA compounds the in vivo activity of these compounds was next assessed. These studies were also designed to provide preliminary data on the oral bioavailability of this class of compounds.

While previous studies with L-CYS-1 (Table 3.1) have demonstrated that this compound is orally bioavailable in rats (Glenn et al., 2004), no data was available for the 2-ASA compounds.

When tested in mice 2-ASA-14 was orally bioavailable with a Cmax and Tmax of 340ng/mL

(720nM) and 1 h, respectively (Figure 3.2). This Cmax is well above the IC50 value achieved in vitro against P. falciparum parasites (13 – 33nM). While these data suggest that appropriate antimalarial plasma levels of 2-ASA-14 can be achieved in vivo, these oral bioavailability results also indicate that this compound is rapidly cleared in mice (Figure 3.2). While pharmacokinetic data are not available for 2-ASA-9 and 2-ASA-13, the poor in vivo activity of these compounds in mice is likely to be due to a short half life. It may be possible to improve antimalarial activity of 2-ASA compounds by increasing the therapeutic dose, however this was not able to be tested in this study due to animal ethics limitations and available information toxicity and tolerability of hydroxamate compounds. More recently in vivo studies have shown that another hydroxamate-based HDAC inhibitor, WR301801, given orally at doses of

50mg/kg/day for four days produced no toxicity in mice and cures were obtained in mice infected with P. berghei in a prophylactic mode (Agbor-Enoh et al., 2009). Likewise, Dow et al., (2008) showed that combination of sub-curative amounts of chloroquine (64mg/kg/day) and

54mg/kd/day of WR301801, once daily for three days, was also able to cure mice infected with

P. berghei in a therapeutic mode. This observation is encouraging as it indicates that HDAC inhibitors may be useful when combined with some antimalarial drugs such as chloroquine. 49

Chapter 3

While future studies on the 2-ASA class of compounds will investigate higher dosing regimens, the poor in vivo half lives and stability of hydroxamates is emerging as an issue that needs to be addressed in the next generation of compounds. Recent work shows that the HDAC inhibitor

WR301801 is metabolised rapidly by mouse and human liver microsomes to produce an ~15 atomic mass unit metabolite that is less active than WR301801. The metabolite displays significantly reduced in vitro antimalarial activity (IC50 >1000nM compared to WR301801 IC50

0.5nM) (Dow et al., 2008). The proposed mechanism for the metabolism of this compound is hydrolysis of the hydroxamic acid zinc binding group (Dow et al., 2008). Preliminary in vitro metabolic studies with 2-ASA-9 also support these findings, demonstrating the existence of a major metabolite ~15 atomic mass less than 2-ASA-9 (Personal communication, Dr. Geoff

Dow, WRAIR). These findings underscore the need to develop new HDAC inhibitors with more desirable drug-like properties such as improved metabolic stability. The search for new zinc binding group will be challenging. A potential issue is the predicted narrow active site in

PfHDAC1 that is almost identical to that found in human HDACs. Therefore, the choice of zinc binding groups to replace the hydroxamic acid group is limited. Previous studies by Colletti et al., (2001a) have shown that replacement of the metal chelating group to the apicidin structure

(to either α-mono-fluoroketone, α-hydroxy-ketone or α-ketoepoxide) can enhance HDAC activity of these compounds. These observations suggest that replacement of the hydroxamate may be possible and this currently the focus of medicinal and structural chemistry.

In vitro drug combination studies are often used to examine potential mode of action of antimalarial compounds and to provide insight into possible useful in vivo combinations. The recommended treatment for uncomplicated falciparum malaria is combination therapy which is designed to reduce the chance of antimalarial drug resistance developing to a monotherapy

(WHO, 2006). For example, atovaquone and proguanil (Malarone) behave synergistically

(Canfield et al., 1995) and together they were use in combination as a chemoprophylaxis and therapeutic treatment for malaria (Hogh et al., 2000). In this study, in vitro isobologram analysis was used to investigate the interaction of SAHA and 2-ASA-9 with the antimalarial drugs 50

Chapter 3 chloroquine, quinine or artemisinin. Combinations of 2-ASA-9 with either chloroquine (I = -

4.44 and P<0.0001; Figure 6.8B) or artemisinin (I = -0.92, P<0.005; Figure 6.8E) were antagonistic. These findings are similar to those reported by Dow et al., (2008) who also observed antagonistic interactions for the experimental HDAC inhibitor WR301801 and the antimalarial drugs chloroquine, azithromycin, mefloquine and artemisinin. It is difficult to ascertain why HDAC inhibitors would be behaving antagonistically with chloroquine as this drug is believed to act in the digestive vacuole of P. falciparum parasites. However, other mechanisms of action may be involved. For example, chloroquine has been shown to bind to

DNA (Marquez et al., 1974) and might interfere with the access of HDAC inhibitors to their target. However, as discussed above studies by Dow et al., (2008) observed cures in mice treated with a combination of chloroquine and WR301801. Taken together, it would seem that in vivo factors may be modulating the interactions between chloroquine and WR301801.

The antagonistic interaction 2-ASA-9 with artemisinin is also difficult to explain but may be due to this drug’s hypothesized action against P. falciparum sarcoplasmic/endoplasmic reticulum calcium ATPase (PfSERCA, Plasmodium genome resource gene ID: PFA0310c)

(Uhlemann et al., 2005). A preliminary microarray study on the effect of HDAC inhibitors on P. falciparum transcription found that PfSerca is down-regulated in 2-ASA-9 treated parasites

(Personal communication, Dr. Kathy Andrews, QIMR (Hu et al., 2009)). While, these findings indicated that HDAC inhibitor may modulate the expression of PfSerca, the effect at the protein level remains unknown.

While the interaction of QNE with SAHA or 2-ASA-9 appears antagonistic, statistical analysis suggests that the “I” value for these combinations crosses unity and as a result that these combinations are likely to be additive (Figure 6.8C and D, P = 0.1). These data suggest that

HDAC inhibitors and quinine have unrelated modes of action and assuming that there are no dangerous pharmacokinetic interactions, might be an interesting combination to further investigate.

Chapter 3 In terms of predicted outcomes of combining antimalarial drugs and HDAC inhibitors in vivo, significant pharmacokinetic interactions are not predicted as most antimalarial drugs are metabolized by cytochrome P450 enzymes (e.g., artemisinin is metabolized by CYP 3A4

(Svensson & Ashton, 1999)). Published data showed that HDAC inhibitors such as SAHA do not inhibit CYP drug metabolizing enzymes in humans (FDA, 2006). Likewise, as discussed above, a metabolite of WR301801 produced in mouse and human liver microsomes was observed in the absence of microsomal factors, indicating that WR301801 metabolism was not cytochrome P450 mediated (Dow et al., 2008).

Chapter 4

Effects of hydroxamate-based HDAC inhibitors on P. falciparum

parasites

Publications arising from this work:

1. Andrews KT, Tran TN*, Lucke AJ, Kahnberg P, Le GT, Boyle GM, Gardiner DL, Skinner-

Adams TS, Fairlie DP: Potent antimalarial activity of histone deacetylase inhibitor analogues.

Antimicrobial Agents and Chemotherapy 2008, 52(4):1454-1461.

*Co-first Author: Equal contribution to manuscript

2. Dow GS, Chen Y, Andrews KT, Caridha D, Gerena L, Gettayacamin M, Johnson J, Li Q,

Melendez V, Obaldia N, 3rd, Tran TN, Kozikowski AP: Antimalarial activity of phenylthiazolyl-bearing hydroxamate-based histone deacetylase inhibitors. Antimicrobial

Agents and Chemotherapy 2008, 52(10):3467-3477.

Chapter 4

4.1 Introduction

As discussed in the Chapter 3, HDAC inhibitors may be useful new antimalarial agents, however most studies involving these compounds have focused on examining the structure activity relationships of different HDAC inhibitors against P. falciparum infected erythrocytes in vitro. There are limited data available that describe the modes of actions of these agents.

Without this information drug action cannot be understood and optimized. In this study, the in vitro pharmacodynamic actions of different HDAC inhibitors against asexual stage P. falciparum parasites were investigated to provide insights into the modes of action of these compounds against malaria parasites. It was also hoped that these studies would provide clues as to the role of PfHDACs in P. falciparum parasites.

4.2 Materials and methods

4.2.1 Stage specific growth inhibition assays

The effect of HDAC inhibitors on the growth of parasites over multiple invasion cycles was determined by microscopic examination of Giemsa-stained thin blood smears. Briefly, synchronous ring (~18 h post invasion), trophozoite (~26 h post invasion) or schizont-stage

(~38 h post invasion) Dd2-infected erythrocytes (starting parasitemia 0.2–1%) were cultured over 4 days under standard culture conditions in the presence of DMSO (0.05%) or test compounds. Percent parasitemia was determined by counting at least 1000 erythrocytes (two independent persons) at each time point and parasitemias were compared to matched DMSO- treated control parasites. Three independent experiments were carried out (representative results are shown). Mean parasitemias at 48h for each treatment group were analysed using analysis of variance (ANOVA) with the experimental replicate included as a covariate. Significance was determined to be a P value of <0.01.

The effect of selected hydroxamate-based HDAC inhibitors on the growth of different P. falciparum intra-erythrocytic stages was assessed using an isotopic micro test (Andrews et al.,

2000). Synchronous Dd2 cultures containing ring- or trophozoite-stage infected erythrocytes at

Chapter 4

0.4% parasitemia and 5% haematocrit were prepared using standard in vitro culture techniques

(Section 2.1). These parasites were exposed to 1×, 5×, or 10× IC50 values of each inhibitor or

DMSO vehicle control (0.05%). After 2, 4, or 6 h compounds were removed by washing with

10 volumes of culture media. A 100µL aliquot of culture was then transferred into triplicate wells of a 96-well micro titre plate which was then labeled with 0.5µCi/well 3H-hypoxanthine

(Amersham, GE Healthcare, UK). Additional control cultures treated with 1× IC50 of each compound were also plated as above, but the compound was not washed off. Plated cultures were then grown under standard culture conditions for 48 h and harvested onto MicroBeta1450 filter-mats (Wallac, Perkin-Elmer, USA). Growth was calculated by the relative amount of hypoxanthine incorporation of treated parasites compared to DMSO vehicle treated controls.

Statistical significance between treated parasites and controls was determined by a two tailed

Student t-test, for 4 – 5 independent experiments.

4.2.2 Transmission electron microscopy (TEM) of HDAC inhibitor treated

parasites

The effect of compounds on the ultra-structure of P. falciparum infected erythrocytes was investigated using transmission electron microscopy (TEM) imaging. Briefly, trophozoite stage

P. falciparum 3D7-infected erythrocytes were cultured under normal conditions in the presence of 10× IC50 values of compounds for 3.5 h. Matched control parasites were cultured in vehicle alone (0.04% DMSO). Infected erythrocytes were then washed three times in 1× PBS (~10 volumes) prior to fixing in freshly prepared fixing solution (4% glutaraldehyde (ProSciTech,

Australia) in 0.1% sodium cocadylate buffer (ProSciTech, Australia), pH 7.4 and adjusted to

300 milli-osmoles with sucrose (BDH, England) and calcium chloride (BDH, England)).

Samples were processed according to standard methods overnight at 4oC (Glauert, 1974).

Briefly, this involved post-fixation with 1% osmium tetroxide (ProSciTech, Australia) and 1% uranyl acetate (Ajax Chemicals, Australia), prior to dehydration in an ascending series of ethanol concentrations. After embedding in Spurr low viscosity resin (ProSciTech, Australia), ultrathin sections (~50 – 60nm thick) were prepared, mounted on 200mesh copper TEM grids,

Chapter 4 and stained with uranyl acetate and lead citrate. Ultrathin sections were examined and photographed with a JEOL 1200EX transmission electron microscope operative at 80kV

(fixation, preparation of ultrathin sections and TEM imaging was carried out by Dr Deb Stenzel at Queensland University of Technology).

4.2.3 Histone hyperacetylation assays

Late-trophozoite-stage P. falciparum Dd2-infected erythrocytes (1 – 2 × 108 parasites/treatment group) were cultured in the presence of compound or DMSO (0.01 – 0.1%) under standard culture conditions for 3.5 h (Section 2.1). Parasites were harvested by saponin lysis (Section

2.7) then washed thoroughly three times in ice cold 1× PBS to remove excess heamoglobin.

Histones were extracted in buffer (10mM Hepes pH 7.9, 1.5mM NaCl, 10mM KCl) containing a final concentration of 0.5M hydrochloric acid (HCl). Acid extraction was carried out on ice for 1.5 h with frequent agitation. Acid-insoluble proteins were pelleted (13, 200rpm at 4oC for

10 min) and the acid-soluble protein fraction precipitated in 1 volume of acetone at -200C overnight. Acid-soluble proteins were pelleted by centrifugation as above, then washed once in

500µL acetone and allowed to air dry completely prior to resuspension in 50µL 1× SDS-PAGE sample buffer. Histone extracts (~2 × 107 parasites equivalent per lane) were resolved by 15%

SDS-PAGE and silver stained to confirm equal loading. To detect changes in acetylation patterns following treatment with compounds, SDS-PAGE separated samples were transferred to PVDF (Roche, Germany) and Western blot carried out using anti-tetra-acetyl-H4 histone antibody or anti-di-acetyl-H3 histone antibody (Section 2.11; 1:2000 dilution; Upstate, USA).

Primary antibodies were detected with goat anti-rabbit horseradish peroxidase (Section 2.11;

1:5000 dilution; Zymed, USA). Membranes were incubated in enhanced chemiluminensce

(ECL) solution (GE Healthcare, UK) and detected on autoradiography film (FujiFilm, Japan).

Chapter 4

4.3 Results

4.3.1 Stage specific effect of HDAC inhibitors on P. falciparum growth in vitro

To determine which intra-erythrocytic P. falciparum life cycle stage is most susceptible to hydroxamate-based HDAC inhibitors, the effect of different compounds on rings (~18 h post invasion), trophozoites (~26h post invasion) and schizonts (~38 h post invasion) was examined microscopically over 2 to 4 days. As shown in Figure 4.1, ring-stage parasites exposed to TSA

(200nM; 25×IC50), 2-ASA-9 or 2-ASA14 (50nM; ~1× IC50 and 200nM; ~4×IC50) were unable to develop normally into trophozoites and schizonts compared to parasite controls (0.05%

DMSO), which matured normally over two invasion cycles. Similarly, trophozoite-stage parasites treated with HDAC inhibitors were not able to develop into schizonts capable of producing invasive merozoites (Figure 4.1). When schizont-stage parasites (~38 h post invasion) were treated with TSA, 2-ASA-9 or 2-ASA-14, some rings were detected after 6 – 12 h, but only at relatively low levels compared to those of control cultures (0.2 – 0.6% ring or schizont culture compared to ~2% ring parasites for control cultures; Figure 4.1). In each case

(ring, trophozoite and schizont assays), a statistical analysis was carried out on data collected at

~48 h for three independent experiments, and there was a significant reduction in parasitemia for TSA, 2-ASA-9 and 2-ASA-14 treated groups compared to controls (P <0.001; Figure 4.2 and Figure 4.3, schizonts data not shown).

Chapter 4

A Rings

10 8

6 4

2 0

% Parasitemia start24487284 Hours

Control TSA (200nM) 2-ASA-9 (50nM) 2-ASA-9 (200nM) 2-ASA-14 (50nM) 2-ASA-14 (200nM)

B Trophozoites 10 8 6 4 2 0 % Parasitemia start12244860 Hours Control TSA (200nM) 2-ASA-9 (50nM) 2-ASA-9 (200nM) 2-ASA-14 (50nM) 2-ASA-14 (200nM)

C Schizonts

10 8 6 4 2 0 % Parasitemia start12304272 Hours Control TSA (200nM) 2-ASA-9 (50nM) 2-ASA-9 (200nM) 2-ASA-14 (50nM) 2-ASA-14 (200nM)

Figure 4.1. Effect of HDAC inhibitors on the growth P. falciparum over multiple cycles. Synchronous ring-, trophozoite- or schizont-stage P. falciparum line Dd2-infected erythrocytes were cultured in the presence of TSA (200nM; ~25× IC50), 2-ASA-9 or 2-ASA-14 (50nM (~1.5× IC50) and 200nM (~ 5× IC50). Parasitemia was determined by microscopic examination of Giemsa-stained thin blood smears over 1 – 4 days (representative experiments are shown above). Error bars indicate standard deviations between replicate counts on the same slide.

Chapter 4

A Control B TSA 8 8 6 6 Rings Rings 4 Trophozoites 4 Trophozoites

Parasitemia Schizonts Schizonts 2 2 % % Parasitemia 0 0 024487284 024487284 Hours Hours

C 2-ASA-9 (50nM) D 2-ASA-14 (50nM) 8 8

6 6 Rings Rings 4 Trophozoites 4 Trophozoites Schizonts Schizonts 2 2 % Parasitemia % Parasitemia

0 0 024487284 0 0 48 72 84 Hours Hours

Figure 4.2. Effect of HDAC inhibitors against ring-stage P. falciparum-infected erythrocytes growth. P. falciparum-infected erythrocytes (Dd2) starting at ring stage were untreated (A; control) or grown in the presence of 200 nM TSA (B), or 50nM 2-ASA-9 (C) or 2-ASA-14 (D). Parasitemia was determined by microscopic examination of Giemsa-stained thin blood smears over ~2-4 days (Representative experiments shown). Error bars indicate standard deviation (SD)

A Control B TSA 8 8

6 6 Rings Rings 4 Trophozoites 4 Trophozoites

Parasiitemia Schizonts Schizonts 2 2 % Parasitemia % 0 0 012244860 012244860 Hours Hours

C 2-ASA-9 (50nM) D 2-ASA-14 (50nM) 8 8 6 6 Rings Rings 4 Trophozoites 4 Trophozoites Schizonts Schizonts 2 2 % Parasitemia % Parasitemia 0 0 0 12244860 012244860 Hours Hours

Figure 4.3. Effect of HDAC inhibitors against trophozoite-stage P. falciparum-infected erythrocytes growth. P. falciparum-infected erythrocytes (Dd2) starting at trophozoite stage were untreated (A; control) or grown in the presence of 200 nM TSA (B), or 50nM 2-ASA-9 (C) or 2-ASA-14 (D). Parasitemia was determined by microscopic examination of Giemsa- stained thin blood smears over ~2-4 days (Representative experiments shown). Error bars indicate standard deviation (SD)

Chapter 4

Next, the effect of exposing ring- and trophozoite-stages to HDAC inhibitors for 2, 4 , or 6 h was examined to determine which of these intra-erythrocytic stages is more susceptible and how quickly these inhibitors act. In comparison to matched control cultures that received vehicle alone (0.05% DMSO), a significant reduction in growth was observed when trophozoites were exposed to SAHA, 2-ASA-9 and 2-ASA-14 at 5× (≥40% inhibition; P < 0.01) and 10× (>60% inhibition; P < 0.01) the IC50 values (Figure 4.4) for 4 or 6 h. Only 2-ASA-14 caused a small

(~25%) but statistically significant reduction in the growth of trophozoites (P <0.01) when diluted to its 1× IC50 value for 4 and 6 h (Figure 4.4). In contrast, ring-stage parasite growth was not significantly affected by treatment with 1× IC50 or 5× IC50 concentrations of these compounds (P >0.05; Figure 4.4). Exposing ring-stage parasites to 10× IC50 concentration of compound for 6 h resulted in a small reduction in growth (~20% inhibition; P < 0.05; Figure

4.4). As expected, control ring-stage parasite cultures exposed continually to each compound at their IC50 (determined from assays set up at ring stage (Table 3.2)) resulted in a ~50% growth

(Figure 4.5; P<0.01). Also as expected from results shown in Figure 4.4, trophozoites were more sensitive to IC50 amounts of compounds upon continual exposure (Figure 4.5).

Chapter 4

Rings-SAHA Trophozoites-SAHA 200 200

150 150

100 * * 100 * * % growth % 50 growth % 50 * * 0 0 2h 4h 6h 2h 4h 6h

Rings-2-ASA-9 Trophozoites-2-ASA-9 200 200

150 150

100 ** 100 * * % growth % % growth * 50 50 *

0 0 2h 4h 6h 2h 4h 6h

Rings-2-ASA-14 Trophozoites-2-ASA-14 200 200

150 150 * * 100 * 100 * *

% growth % growth % * 50 50 * * * 0 0 2h 4h 6h 2h 4h 6h

Figure 4.4. Stage-specific effect of HDAC inhibitors on P. falciparum ring and trophozoite- stage infected erythrocytes. Synchronous ring- or trophozoite-stage P. falciparum line Dd2- infected erythrocytes were treated with SAHA, 2-ASA-9 or 2-ASA-14 at either 1× IC50 (blue bars), 5× IC50 (red bars) or 10× IC50 (green bars) for 2 h, 4 h or 6 h followed by washing and assessing parasite growth 48 h after beginning the experiments. Percentage growth (± standard deviation) relative to untreated DMSO controls is shown for 4 – 5 independent assays. Asterisk indicates significant difference compared to untreated controls (P <0.05).

Chapter 4

Rings Trophozoites 100 100

50 * 50 * * * * % growth % growth % *

0 0 SAHA 2-ASA-9 2-ASA-14 SAHA 2-ASA-9 2-ASA-14

Figure 4.5. Effect of continuous exposure of HDAC inhibitors on the growth of P. falciparum ring and trophozoite-stage infected erythrocytes. Synchronous ring- or trophozoite-stage P. falciparum line Dd2-infected erythrocytes were treated with either 1× IC50 SAHA (25nM), 2-ASA-9 (39nM) or 2-ASA-14 (33nM) for 48 h. Percentage growth (± standard deviation) relative to untreated DMSO controls is shown for 4 – 5 independent assays. Asterisk indicates significant difference compared to untreated controls (P <0.05).

4.3.2 Effect of HDAC inhibitor treatment on P. falciparum morphology

As trophozoites were found to be more susceptible to HDAC inhibitors than ring stages, this parasite stage was used for ultrastructural studies using transmission electron microscopy

(TEM) analysis. TEM of trophozoite infected erythrocytes treated with SAHA, 2-ASA-9, and 2-

ASA-14 showed that HDAC inhibitor-treated parasites have a less organized cytoplasm than untreated controls (representative parasite images are shown; Figure 4.6). HDAC inhibitor treated parasites contain large electron-lucent areas in the cytoplasm that appeared to be lipid droplets (white arrows; Figure 4.6). These parasites also often contain abnormal nuclei with dense patches throughout the nucleoplasm (asterisks; Figure 4.6) and some separation of the membranes surrounding organelles so that clear spaces could be observed between the membranes and the organelle matrix. Overall, HDAC inhibitors appear to have a general toxic effect on parasites following 6 h exposure, which correlates with the observation of rapid killing of trophozoite-stage parasites in Section 4.3.1. Control parasites were evenly stained throughout with clearly defined structures (Figure 4.6A). Structures such as digestive vacuole and knobs appear to be unaffected by HDAC inhibitors.

Chapter 4

A B C

Figure 4.6. Effect of HDAC inhibitor treatment on P. falciparum infected erythrocyte morphology. Trophozoite-stage 3D7-infected erythrocytes were treated with 0.05% DMSO (A), SAHA (10× IC50; 250nM, B),or 2-ASA-9 (10× IC50; 390nM, C). White arrows indicate what appears to be lipid droplets, asterisks indicate condensed patches in the parasite nuclei and black arrows indicate knobs on the red cell surface. DV: digestive vacuole and N: nucleus.

Chapter 4

4.3.3 Effect of HDAC inhibitors on P. falciparum histone acetylation status

The effect of HDAC inhibitors on P. falciparum histone acetylation status was examined using trophozoite-stage infected erythrocytes treated with different amounts of TSA, SAHA, 2-ASA-

9, 2-ASA-14, WR301801, or no compound (control), for 3.5 h. Tetra-acetyl-histone H4 antisera recognised a predominant band of ~13kDa in all P. falciparum histone samples and control histones from calf thymus (Sigma, USA). These bands most likely represent the normal histone acetylation state of the organism as the same bands were seen in untreated starting samples (0 h) and the 3.5 h controls (Figure 4.7 and Figure 4.8). The tetra-acetyl-histone H4 antibody also cross-reacted with an ~15kDa protein band which corresponds to the histone H2A.Z species which was previously confirmed by others by mass spectrometry and an antibody raised against acetyl-histone H4 lysine 12 (Miao et al., 2006). A dose dependent effect on the histone H4 pattern was observed for parasites treated with SAHA, 2-ASA-9 and 2-ASA-14, as indicated by a laddered profile and an increase in the intensity of the ~13kDa band (Figure 4.7 and Figure

4.8). Compound WR301801 (Dow et al., 2008), the most potent compound in this panel of antimalarial HDAC inhibitors (with the exception of TSA which was included as a control and tested at 100nM and 500nM), caused hyperacetylation of histone H4 at all concentrations tested

(Figure 4.8). The effect of HDAC inhibitors SAHA and WR301801 on acetylated histone H3 was also tested using a di-acetyl-histone H3 antibody. A single band of ~15kDa was detected in all samples, however the intensity of the signal increased in samples treated with SAHA or

WR301801 (Figure 4.8). No change in H3 or H4 histone acetylation pattern was observed for infected erythrocytes treated with 1× IC50 (1.8nM) artemisinin (Figure 5.5) or 250× IC50

(500nM) artemisinin (data not shown).

Chapter 4

2-ASA-9 2-ASA-14 A B kDa TSA TSA 500nM 500nM Control Control 100nM 20nM 100nM 20nM CT CT 15 – H2A.Z H4 10 – anti-(tetra)acetyl H4

Silver stain

15 – 10 –

Figure 4.7. Effect of 2-ASA compounds on P. falciparum histone acetylation. P. falciparum Dd2-infected erythrocytes were cultured in vitro in the presence of (A) TSA (500nM; 45× IC50), 2-ASA-9 (20nM; ~1× IC50, 100nM; ~5× IC50 and 500nM; 25× IC50) or (B) 2-ASA-14 (20nM; ~1× IC50, 100nM; ~5× IC50 and 500nM; 25× IC50) for 3.5 h. Matched controls received no drug (control; 0.1% DMSO). Histones were acid extracted in 0.5M HCl (Section 4.2.3) and separated via 15% SDS-PAGE gel electrophoresis. Hyperacetylation was determined by Western blotting using polyclonal anti-tetra-acetyl histone H4 antisera (1:2000, Upstate; USA). Silver staining was carried out as a loading control. Arrows indicate different histones. CT; calf thymus histone (Sigma, USA).

SAHA WR301801

kDa Art TSA Control DMSO 2000 nM 100 nM 10 nM 1 nM 1000 nM 100 nM 10 nM 1 nM 15 – H2A.Z 10 – H4 anti-(tetra)acetyl H4

15 – anti-(di)acetyl H3

Silver stain

15 –

10 –

Figure 4.8. Effect of SAHA and WR301801 on P. falciparum histone acetylation. P. falciparum Dd2-infected erythrocytes were cultured in vitro in the presence of TSA (100nM; 12.5× IC50), SAHA (10nM; 1× IC50, 10nM; 1× IC50, 100nM; 4× IC50 and 2000nM; 80× IC50) WR301801 (1nM; 1× IC50, 10nM; 10× IC50, 100nM; 100× IC50 and 1000nM; 1000× IC50) for 3.5 h. Matched controls received no drug (control; start or DMSO; 0.1%) or Artemisinin (Art, 1.8nM; ~1× IC50). Histones were acid extracted in 0.5M HCl (Section 5.2.3) and separated via 15% SDS-PAGE gel electrophoresis. Hyperacetylation was determined by Western blotting using polyclonal anti-tetra-acetyl histone H4 antisera (1:2000, Upstate; USA) and anti-di-acetyl histone H3 antisera (1:2000, Upstate, USA). Silver staining was carried out as a loading control. Arrows indicate different histones. CT; calf thymus histone (Sigma,USA).

Chapter 4

4.3.4 Effect of HDAC inhibitors on deacetylase activity of P. falciparum nuclear

extracts

As discussed in the next chapter (Chapter 5) soluble recombinant PfHDAC1 was able to be expressed in E. coli, however, as this protein was inactive, studies evaluating its function could not be pursued. As an alternative, P. falciparum nuclear extracts were used to determine whether HDAC inhibitors affect parasite deacetylase activity. These studies demonstrated that

TSA, SAHA, 2-ASA-9 and 2-ASA-14 inhibit deacetylase activity in P. falciparum nuclear extracts at nanomolar concentrations and in a dose-dependent manner (Figure 4.9). The IC50 values of TSA, 2-ASA-9 and 2-ASA-14 in these enzyme assays were all below 100nM. The inhibitory effect of SAHA were less potent (this compound is also a less potent antimalarial;

Table 4.1) and more variable (IC50 155nM ± 127nM). At 1µM all HDAC inhibitors reduced deacetylase activity in parasites nuclear extracts to less than 15% of the untreated control.

SAHA TSA 2-ASA-9 2-ASA-14 16000 14000 12000 10000 8000 AFU 6000 4000 2000 0 0 31.25 62.5 125 250 500 1000

Compound (nM)

Figure 4.9. Dose dependent inhibition of P. falciparum deacetylase activity by hydroxamate-based HDAC inhibitors. P. falciparum Dd2-nuclear extracts (~5 × 107 parasites equivalent used in each reaction) were assayed for deacetylase activity in a 96-well micro titre plate using a commercial deacetylase activity kit (Section 2.10; Upstate, USA) and either TSA, SAHA, 2-ASA-9, or 2-ASA-14. Deacetylase activity was measured as arbitrary fluorescence units (AFU) at excitation 370nm and emission 460nm on PolarStar plate reader (BMG). Results are shown for two independent extracts (mean + SEM) prepared on separate days, each tested in duplicate wells.

Chapter 4

Table 4.1. Inhibitory activity of HDAC inhibitors on whole parasites and P. falciparum deacetylase activity. Dd2 IC50 nM (SD) Maximum Compound Whole cell-assay Deacetylase activity deacetylase inhibition (1µM) %b TSA 8a <32nM 84 SAHA 251 (27) 155 (127) 92 2-ASA-9 39 (24) 93 (10) 92 2-ASA-14 33 (33) 78 (3) 91 a IC50 value obtained from (Mai et al., 2004) b HeLa % deacetylase inhibition at 1µM (91 ± 0.1)

4.3.5 HDAC inhibitors alter transcription of alpha-II-tubulin in P. falciparum

parasites

To investigate whether HDAC inhibitors can alter gene expression in P. falciparum parasites,

Northern blot (Section 2.14) was carried out on P. falciparum RNA extracted from parasites exposed to quinine (as a control), SAHA, 2-ASA-9 or 2-ASA-14 at various concentrations for

3.5 h. Parasites treated with DMSO (0.05%) were included as a negative control. The alpha-II- tubulin transcript was shown to be up-regulated in HDAC inhibitor treated samples with an increase in hybridisation intensity at higher concentrations (50nM and 500nM; Figure 4.10). In contrast, alpha-II-tubulin transcript was not detectable in DMSO control or quinine treated samples. The histone H4 transcript remained unchanged in all samples tested, and served as a loading control.

Chapter 4

QNE SAHA 2-ASA-9 2-ASA-14

50 nM 500 nM 50 nM 500 nM 50 nM 500 nM DMSO 500 nM 100 nM

4kb – α-II-tubulin

2kb – Histone H4

Ethidium Bromide

Figure 4.10. Effect of HDAC inhibitor treatment on P. falciparumm alpha-II-tubulin gene transcription. Trophozoite-stage P. falciparum Dd2-infected erythrocytes were treated with quinine (QNE, 100nM; ~2× IC50 and 500nM; 10× IC50) or HDAC inhibitor compounds SAHA (50nM; 2× IC50 and 500nM; 20× IC50), 2-ASA-9 (50nM; ~2× IC50 and 500nM; 10× IC50) or 2- ASA-14 (50nM; ~2× IC50 and 500nM; 10× IC50). Matched control samples received vehicle alone (0.05% DMSO). Total RNA were separated via 0.8% TBE agarose gel electrophoresis and transferred to nitrocellulose membrane. Membrane was sequentially stripped and reprobed with α-II-tubulin then histone H4 specific PCR probes (primer details are listed in Table S3). Ethidium bromide staining of the agarose gel is shown as a loading control. Size in kilobase (kb) is shown.

4.4 Discussion

The effect of hydroxamate-based HDAC inhibitors on P. falciparum was examined to better understand when and how these compounds act on malaria parasites. Stage specific growth inhibition assays using TSA, SAHA, 2-ASA-9 and 2-ASA-14 show that these HDAC inhibitors all inhibit the growth of rings, trophozoites and schizonts over long treatment times (>48 h).

When short treatment times were assessed, the HDAC inhibitors SAHA, 2-ASA-9 and 2-ASA-

14 were found to preferentially inhibit the growth of trophozoites as compared to rings and this effect was rapid (within 4 – 6 h of treatment). These stage specific growth inhibition activity data are in line with the expression of PfHDAC1 protein which peaks during the trophozoite-

Chapter 4 stage of the intra-erythrocytic developmental cycle (Section 5.3.5). These in vitro effects may also explain, in part, the relatively poor in vivo antimalarial activity of 2-ASA compounds observed in Chapter 3. If 2-ASA compounds target trophozoites but not rings, in vivo studies with short half life compounds in a mouse model that produces a synchronous infection, would need to ensure that dosing is timed correctly.

To further investigate the effect of hydroxamate-based HDAC inhibitors on P. falciparum parasites transmission electron microscopy was carried out to examine morphological effects at the ultra-structural level. Nuclei in trophozoites appear to have dense patches (asterisks; Figure

4.6) following treatment with HDAC inhibitors for 6 h indicating these compounds might be affecting chromatin integrity in parasites. Previous studies have shown that mammalian cells treated with the HDAC inhibitor TSA display significant changes in chromatin distribution with reduced heterochromatin at the nuclear periphery (Galiova et al., 2008). Although, the effect of

HDAC inhibitors on parasite nuclear integrity will need further examination these results are not surprising given the effect of HDAC inhibitors on parasite histone acetylation state.

Histone hyperacetylation is a marker of HDAC inhibition commonly used to investigate HDAC inhibitors in cancer studies. Like the HDAC inhibitors apicidin and TSA in this study, a panel of hydroxamate-based HDAC inhibitors (TSA, SAHA, 2-ASA-9, 2-ASA-14 and WR301801) was shown to cause hyperacetylation of parasite histones in a dose dependent manner using anti- tetra acetyl H4 antibodies. While a radiometric assay has been previously used to show that apicidin hyperacetylates P. falciparum histones (Darkin-Rattray et al., 1996), the method described here is the first use of Western blot and commercial anti-histone antisera to study histone hyperacetylation in malaria parasites. This assay is simple, robust, and does not require radioactivity and is therefore a good tool for mode of action assays in the development of

HDAC inhibitors for malaria. In addition to examining parasite acetylation patterns in response to HDAC inhibitor treatment, a new approach using parasite nuclear extracts was used to confirm that HDAC inhibitors also inhibit P. falciparum deacetylase activity. TSA, SAHA, 2-

Chapter 4

ASA-9 and 2-ASA-14 all inhibited deacetylase activity in nuclear extracts in a dose dependent manner with IC50 values below 100nM (with the exception of SAHA - IC50 =155nM). In addition, at the highest test concentration (1µM) the deacetylase activity in the parasite nucleus at least able to be detected using this commercial kit, is almost completely inhibited (>90%).

Together these acetylation and enzyme activity data indicate that HDAC inhibitors are inhibiting PfHDAC activity in the parasite.

In an effort to understand more about the effect of HDAC inhibitors on P. falciparum transcription, a preliminary microarray study of the effect of TSA on P. falciparum gene transcription was carried out in parallel to this work (Dr. Kathy Andrews, QIMR and Prof.

Zbynek Bozdech, NTU, Singapore (Hu et al., 2009)). Preliminary data from this study suggest that the transcription of up to 10% of genes is changed as a result of HDAC inhibitor treatment.

To confirm these preliminary results, one of these genes, alpha-II-tubulin, was selected for

Northern blot studies. Alpha-II-tubulin is normally only present at low levels in asexual stage parasite, as previously reported (Delves et al., 1990). Trophozoites treated with the HDAC inhibitors SAHA, 2-ASA-9 and 2-ASA-14 (50nM and 500nM) cause an increase in alpha-II- tubulin hybridization signal intensity. Very low levels of alpha-II-tubulin transcript signal were detected in untreated control parasites and parasites treated with a control antimalarial drug, quinine. The transcription profile of histone H4 was not affected by HDAC inhibitor treatment, as predicted based on microarray data which show this gene was unaffected (personal communication, Dr. Kathy Andrews). These findings indicate that PfHDAC activity targeted by

HDAC inhibitors regulate the expression of alpha II tubulin in parasites. In addition, this study also demonstrates the potential of microarray data to provide hints on downstream effects of

HDAC inhibition in malaria parasites. It will be interesting in future studies to determine if parasite-specific transcriptional changes can be identified as these genes may be used as biomarkers to screen for new parasite-selective compounds. Taken together, data presented in this Chapter has contributed to our understanding of the mode of action of HDAC inhibitors in

Chapter 4

P. falciparum parasites and provided new tools for understanding parasite biology and the development of new compounds to target malaria.

Chapter 5

Characterisation of P. falciparum histone deacetylase 1

Publication arising from this work:

Andrews KT, Tran TN*, Lucke AJ, Kahnberg P, Le GT, Boyle GM, Gardiner DL, Skinner-

Adams TS, Fairlie DP: Potent antimalarial activity of histone deacetylase inhibitor analogues.

Antimicrobial Agents and Chemotherapy 2008, 52(4):1454-1461.

*Co-first Author: Equal contribution to manuscript

Chapter 5

5.1 Introduction

Histone deacetylases (HDACs) are members of a large family of eukaryotic proteins. In humans, HDAC enzymes are divided into four major groups based on their sequence structure, subcellular localisation and mechanism of deacetylation. Class I and II enzymes are similar in that they both contain a zinc dependent catalytic core. The class I enzymes are homologues to yeast Rpd3 and are predominantly localised to the cell nucleus (de Ruijter et al., 2003). Class II enzymes differ to class I in that they are larger, display tissue specific expression profiles and can be shuttled between the cell cytoplasm and nucleus (reviewed in Bertos et al., 2001 and references therein). Class III or sirtuin family HDACs are significantly different to their class

I/II relatives in that they require nicotinamide adenosine dinucleotide (NAD) for activity (Dali-

Youcef et al., 2007). In humans, the class IV HDAC, HDAC 11, is localised to the cell nucleus and also displays tissue specific expression similar to the class II HDACs.

The P. falciparum genome contains five putative HDAC encoding genes, including one class I homologue (Plasmodium Genome Resource gene ID: PFI1260c) also referred to as PfHDAC1, two class II homologues (PF14_0690 and PF10_0078) and two class III homologues

(PF13_0152 and PF14_0890). The two class III homologues are not essential to P. falciparum growth in vitro (Freitas-Junior et al., 2005, Tonkin et al., 2009), and only limited information is available about PfHDAC1 or the two class II homologues (Joshi et al., 1999). PfHDAC1 has been shown to localise to the parasite nucleus and is expressed in trophozoites, schizonts and gametocytes (Joshi et al., 1999). Little is known about the role of this protein in P. falciparum parasites, but it has been proposed as the target of class I/II HDAC inhibitors (Joshi et al.,

1999). The aim of this study was to investigate the similarity of this PfHDAC to other eukaryotic HDACs and determine the stage specific expression of the protein in the intra- erythrocytic parasites and to try to express recombinant PfHDAC1 protein.

Chapter 5

5.2 Materials and methods

5.2.1 Sequence alignment and construction of phylogenetic trees

Amino acid sequence searches of class I HDAC homologues were carried out using the ApiDB

(www.ApiDB.org) and GenBank (www.ncbi.nlm.nih.gov) databases. Full length sequences were retrieved and multiple sequence alignment carried out using clustalW (Thompson et al.,

1994). Phylogenetic analysis was performed, with no correction for multiple substitutions on the conserved core, for amino acid residues 4 – 377 of PfHDAC1 (Genbank accession: CAD51938) using clustalX software (Thompson et al., 1997). Phylogenetic trees were constructed using bootstrapped (1000 trials) neighbour joining analysis and trees were constructed using Clustal

X2 (Larkin et al., 2007) and visualized using FigTree v1.1.2 (Rambaut, 2008). Multiple sequence alignment was also carried out on the full length amino acid sequences of P. falciparum class II homologues.

5.2.2 Generation of an in silico PfHDAC1 homology model

The homology model of PfHDAC1 was generated in collaboration with Dr Andrew Lucke and

Professor David Fairlie (Institute for Molecular Bioscience, UQ). Methods are listed in the attached published manuscript (Andrews et al., 2008).

5.2.3 Generation of PfHDAC1 polyclonal antisera

A peptide corresponding to residues 428-448 of PfHDAC1 (Joshi et al., 1999) was synthesized and conjugated to diphtheria toxin (DT) in-house at the QIMR peptide facility. This peptide was administered to BALB/c mice using standard immunization regimens (Gardiner et al., 2004).

Each mouse received 30µg peptide-DT dissolved in 50µL PBS and emulsified with 50µL

Complete Freund’s Adjuvant (Sigma, USA) (100µL final volume) subcutaneously at the tail base. Boosts 30µg peptide-DT dissolved in dissolved in 50µL PBS and emulsified with 50µL

Incomplete Freund’s Adjuvant (Sigma, USA) (100µL final volume) were administered via intra-peritoneal injection three weeks after the first injection, then every second week thereafter for four weeks. A 10µL volume of blood was collected from each mouse at the beginning of the

Chapter 5 immunisation regimen (pre-immune sera) and before each booster injection. Sera were recovered from bleeds and diluted 1:20 with PBS and stored at -200C. All sera were tested for reactivity against total P. falciparum protein extracts using standard Western blot methods

(Section 2.11). For final bleed, mice were euthanised and blood collected via heart puncture.

The blood (1-2 mL per mouse) was allowed to clot overnight at 40C and serum separated by centrifugation at 5000rpm for 30 min. Undiluted sera were aliquoted and stored at -200C.

Antibody titre for use in Western blot was determined using P. falciparum total protein extracts from asynchronous cultures (Figure 5.1). Antibody dilutions of 1:1000, 1:2000, and 1:5000 were examined and 1:1000 or 1:2000 dilutions selected for Western blotting experiments.

Pre-immune

1:1000 1:2000 kDa 70- 55-

43-

Figure 5.1 Analysis of anti-PfHDAC1 polyclonal antisera. D10 P. falciparum total protein extracts (~1 × 106 parasites/lane) were separated via 12% SDS-PAGE gel electrophoresis and transferred to PVDF membrane. Membranes were probed with either pre-immune (1:500), or 1:1000 and 1:2000 dilution of anti-PfHDAC1 antisera. A band of the expected size of PfHDAC1 (51.3kDa) was detected (arrow). Molecular size in kiloDaltons (kDa) is shown.

5.2.4 Preparation of RNA and protein for expression profiling

P. falciparum infected erythrocytes (3D7) were grown under standard culture conditions

(Section 2.1) and synchonised by sorbitol treatment (Section 2.6) to enrich for ring-stage parasites. Parasite samples for RNA and protein were collected starting at very early ring-stage and every six hour thereafter for ~48 h (equivalent to one complete asexual developmental cycle). Parasite pellets are recovered following saponin lysis and washing in PBS (Section 2.7).

The recovered parasite pellets (~1 × 108 parasites) were resuspended in 1mL TRIzol solution

Chapter 5

(Invitrogen, USA) and RNA was prepared using standard methods (Section 2.13). RNA (~1 ×

107 parasites/lane) was separated via 0.8% agarose gel electrophoresis and transferred to nitrocellulose membrane (Amersham, UK). Northern blots were carried out using standard methods (Section 2.14). Alternatively, for protein extracts P. falciparum parasite pellets were resuspended in 100µL 1× SDS-PAGE sample buffer, heat denatured at 900C for 3 min, centrifuged briefly (1 min) and the soluble protein fraction separated via 12% SDS-PAGE gel electrophoresis and transferred to PVDF membrane (Roche, Germany). Western blot was carried out using standard methods with anti-PfHDAC1 (1:1000 dilution), anti-PfAMA-1

(1:3000 dilution) and anti-PfHSP70 (1:10000 dilution) antibodies (Section 2.11; Table S2).

5.2.5 Cloning and purification of PfHDAC1

The full-length Pfhdac1 gene, minus the stop codon, was PCR amplified from 3D7 genomic

DNA using standard PCR conditions and the following primers: h1BAMHI_f

5’CGGgatccgATGTCTAATAGAAAAAAGGTTGC3’ and h1HINDIII_r

5’CCCaagcttTTAATATGGTACAATAGATTGATCC3’ (Section 2.16). PCR products were cloned into the pGEM-T vector (Promega, USA) and representative clones sequenced to confirm that the correct sequence was present and in frame. The Pfhdac1 fragment was excised using BamHI and HindIII restriction enzymes (New England Biolabs, UK) and subcloned into the pRSET-6×His E. coli expression vector (Invitrogen, USA). Resulting pRSET-6×His-

PfHDAC1 plasmid was co-transformed with the pRIG plasmid (Baca & Hol, 2000) (plasmid encodes for tRNAs: Arg, Ile, Gly) into BL21 E. coli cells. A pilot expression study was carried out on a selected clone to determine optimal expression under different induction conditions with isopropyl β-D-1-thiogalactopyranoside (IPTG) induction and expression confirmed using

Western blot with anti-PfHDAC1 (1:1000 dilution) and anti-RGS-His (1:1000 dilution; Qiagen,

USA) antibodies (Section 2.11). 6×His-PfHDAC1 protein was extracted from E.coli cells in

1/10 of volume of the original culture in buffer containing 1× protease inhibitor cocktail (Roche,

Germany) and 1mM imidazole (Sigma, USA) by ~3 cycles of freeze/thaw in dry ice-ethanol bath (10 min) and 370C water bath. Briefly, recombinant 6×His-PfHDAC1 protein was purified

Chapter 5 by batch binding on cobalt affinity resin (Talon, Clontech, USA). Insoluble debris were removed by centrifugation at 13200rpm for 30 min and the soluble protein fraction was transferred to a fresh tube containing 1/10 bed volume of pre-washed (three washes with 10 volume of binding buffer) cobalt affinity resin and allowed to bind overnight at 40C, end-over- end. Unbound supernatant was removed by pelleting the cobalt resin at 2000rpm for 1 min. The resin was then washed three times in 10 volumes of wash buffer containing 10mM imidazole.

Proteins were eluted three times in one volume of elution buffer containing 150mM imidazole.

Purified proteins were analysed by Western blot (Section 2.11) using anti-PfHDAC1 antibody.

5.3 Results

5.3.1 Phylogenetic analysis of the P. falciparum class I HDAC, PfHDAC1

The similarity of PfHDAC1 to other Plasmodium and eukaryotic HDACs was compared. A detailed phylogenetic analysis was carried out including comparison with other Plasmodium species and medically important apicomplexan parasites. All Plasmodium parasites contain a class I zinc-dependent HDAC homologue. These proteins are highly conserved in all plasmodium species (>90% sequence identity; Figure S1). Phylogenetic analysis also demonstrates that these homologues are closely related and form a tight cluster that is closely related to Toxoplasma gondii and Cryptosporidium homologues (shown in yellow; Figure 5.2).

PfHDAC1 shares ~73% sequence identity with the T. gondii homologue (TgHDAC3; Figure

5.2) and ~65% sequence identity with Crytosporidium homologues (ChRpd3B and CpRpd3B;

Figure 5.2).

Chapter 5

S. cerevisiae ScRpd3 [AAB20328]

1000 C. hominis ChRpd3A [XP_668169] 567 C. parvum CpRpd3A [XP_627509] 340 H. sapiens HsHDAC3 [NP_003874]

1000 H. sapiens HsHDAC1 [CAG46518]

965 M. musculus MmHDAC1 [AAI08372]

567 1000 D. rerio DrHDAC1 [AAI65208] 1000 H. sapiens HsHDAC2 [NP_001518]

D. melanogaster DmRpd3 [NP_647918]

C. hominis ChRpd3B [XP_667698] 1000 1000 C. parvum CpRpd3B [XP_625348] 917 T. gondii TgHDAC3 [AAY53803]

1000 P. falciparum PfHDAC1 [CAD51938] 835 P. berghei PbHDAC1 [CAH98271] 356 932 P. yoelii PyHDAC1 [EAA15658]

P. chabaudi PcHDAC1 [CAH87890] 551 P. vivax PvHDAC1 [XP_601614790]

P. knowlesi PkHDAC1 [XP_002258527]

1000 H. sapiens HsHDAC8 [AAF73428] A. aeolicus AaHDLP [NP_213698] 0.1

Figure 5.2. Phylogenetic relationship of selected class I HDAC homologues. Phylogenetic analysis was performed with no correction for multiple substitutions on the conserved deacetylase domain corresponding to amino acid residues 4 – 377 of PfHDAC1 (Genbank accession number: CAD51938) using the clustalX2 software (Thompson et al., 1997). Phylogenetic trees were constructed using bootstrapped (1000 trials) neighbour joining. Bootstrap values approaching 1000 indicate highest confidence. Nomenclature: Organism name, gene name and [accession number] shown on tree. 10% divergent scale bar is shown.

5.3.2 Sequence analysis of PfHDAC1

The N-terminal region of Plasmodium class I HDACs consists of a core deacetylase domain

(300 - 390 amino acids) which is well conserved in virtually all class I members. The deacetylase core regions of Plasmodium class I HDAC homologues consist of the seven signature motifs (motif I-VII; Figure 5.3) that define the acetoin utilization protein/acetylpolyamine aminohydrolase protein superfamily (Leipe & Landsman, 1997).

Catalytically important residues are marked with asterisks in Figure 5.3. Interestingly, all apicomplexan class I homologues contain a two amino acid insertion (always AT, with exception of TT in Cryptosporidium, shown in yellow; Figure 5.3,) corresponding to amino acids 95-96 of PfHDAC1. This insertion is absent in all other eukaryotic class I members except for the bacterial HDLP which has a tyrosine (Y) in the position corresponding residue 96 of

PfHDAC1 (Figure S1).

Chapter 5

Motif I PfHDAC1 ------MSNRKKVAYFHDPDIGSYYYGAGHPMKPQRIRMTHSLIVSYNLYKY 46 TgHDAC3 ------MALSALRKRVAYFYDPDIGSYYYGPGHPMKPQRIRMAHALVLSYDLYKH 49 CpRpd3 ILLVSHLNNIVVISLKMAKRVSYFYDGDIGSYYYGPGHPMKPQRIRMAHNLILSYDLYKH 60 HsHDAC1 ------MAQTQGTRRKVCYYYDGDVGNYYYGQGHPMKPHRIRMTHNLLLNYGLYRK 50 MmHDAC1 ------MAQTQGTKRKVCYYYDGDVGNYYYGQGHPMKPHRIRMTHNLLLNYGLYRK 50 *

Motif II Motif III PfHDAC1 MEVYRPHKSDVNELTLFHDYEYIDFLSSISLENYREFTYQLKRFNVGEATDCPVFDGLFQ 106 TgHDAC3 MEVYRPHKSIEPELCLFHSSDYISFLSSVSPENYKEFSLQLKNFNVGEATDCPVFDGLFT 109 CpRpd3 MEIYKPHKSPQSELVYFHEEDYINFLSSINPDNSKDFGLQLKRFNLGETTDCPVFDGLFE 120 HsHDAC1 MEIYRPHKANAEEMTKYHSDDYIKFLRSIRPDNMSEYSKQMQRFNVGE—-DCPVFDGLFE 108 MmHDAC1 MEIYRPHKANAEEMTKYHSDDYIKFLRSIRPDNMSEYSKQMQRFNVGE—-DCPVFDGLFE 108 *

Motif IV Motif V PfHDAC1 FQQSCAGASIDGASKLNHHCADICVNWSGGLHHAKMSEASGFCYINDIVLGILELLKYHA 166 TgHDAC3 FQQACAGASIDAAKKLNHHQADICVNWSGGLHHAKRSEASGFCYINDIVLGILELLKYHA 169 CpRpd3 FQQICAGGSIDGAYKLNNEQSDICINWSGGLHHAKRSEASGFCYINDIVLGILELLKYHA 180 HsHDAC1 FCQLSTGGSVASAVKLNKQQTDIAVNWAGGLHHAKKSEASGFCYVNDIVLAILELLKYHQ 168 MmHDAC1 FCQLSTGGSVASAVKLNKQQTDIAVNWAGGLHHAKKSEASGFCYVNDIVLAILELLKYHQ 168 **

Motif VI PfHDAC1 RVMYIDIDVHHGDGVEEAFYVTHRVMTVSFHKFGDYFPGTGDITDVGVNHGKYYSVNVPL 226 TgHDAC3 RVMYIDIDIHHGDGVEEAFYVSHRVMTVSFHKFGDFFPGTGDVTDVGASQGKYYAVNVPL 229 CpRpd3 RVMYIDIDVHHGDGVEEAFYLSHRVLTVSFHKFGEFFPGTGDITDIGVAQGKYYSVNVPL 240 HsHDAC1 RVLYIDIDIHHGDGVEEAFYTTDRVMTVSFHKYGEYFPGTGDLRDIGAGKGKYYAVNYPL 228 MmHDAC1 RVLYIDIDIHHGDGVEEAFYTTDRVMTVSFHKYGEYFPGTGDLRDIGAGKGKYYAVNYPL 228 * **

Motif VII PfHDAC1 NDGMTDDAFVDLFKVVIDKCVQTYRPGAIIIQCGADSLTGDRLGRFNLTIKGHARCVEHV 286 TgHDAC3 NDGMDDDSFVALFKPVITKCVDVYRPGAIVLQCGADSLTGDRLGKFNLTIKGHAACVAFV 289 CpRpd3 NDGIDDDSFLSLFKPIISKCIEVYRPGAIVLQCGADSVRGDRLGRFNLSIKGHAECVEFC 300 HsHDAC1 RDGIDDESYEAIFKPVMSKVMEMFQPSAVVLQCGSDSLSGDRLGCFNLTIKGHAKCVEFV 288 MmHDAC1 RDGIDDESYEAIFKPVMSKVMEMFQPSAVVLQCGSDSLSGDRLGCFNLTIKGHAKCVEFV 288

PfHDAC1 RSYNIPLLVLGGGGYTIRNVSRCWAYETGVVLNKHHEMPDQISLNDYYDYYAPDFQLHLQ 346 TgHDAC3 KSLDIPLLVLGGGGYTIRNVARCWAYETGVVLDRHREMSPHVPLNDYYDYYAPDFQLHLT 349 CpRpd3 KKFNIPLLILGGGGYTIRNVARTWAYETATILDRTDLISDNIPLNDYYDYFAPDFKLHIP 360 HsHDAC1 KSFNLPMLMLGGGGYTIRNVARCWTYETAVALD—-TEIPNELPYNDYFEYFGPDFKLHIS 346 MmHDAC1 KSFNLPMLMLGGGGYTIRNVARCWTYETAVALD—-TEIPNELPYNDYFEYFGPDFKLHIS 346

PfHDAC1 PSNIPNYNSPEHLSRIKMKIAENLRHIEHAPGVQFSYVPPDFFNSDID----DESDK--- 399 TgHDAC3 PSSIPNSNSPEHLEKIKTRVLSNLSYLEHAPGVQFAYVPPDFFGEDND----DEDEF--- 402 CpRpd3 PLNLPNMNSPEHLEKIKAKVIDNLRYLEHAPGVEFAYVPSDFFDREASNLQKQEDEE--- 417 HsHDAC1 PSNMTNQNTNEYLEKIKQRLFENLRMLPHAPGVQMQAIPEDAIPEESGDEDEDDPDKRIS 406 MmHDAC1 PSNMTNQNTNEYLEKIKQRLFENLRMLPHAPGVQMQAIPEDAIPEESGDEDEEDPDKRIS 406

PfHDAC1 ------NQYELKDDS---GGGRAPGTRAKEHS-TTHHLRRKNYDDDFFDLSDRDQSI 446 TgHDAC3 ------MQNQVDNE----GGGRAAGATAHTAANAPYRIRRKDYANDFEDMADRDQ-K 448 CpRpd3 ------REEELSSWQ---GGGRAAGSTESQGN---HNEKPKSSRKLQKEHASEFY—460 HsHDAC1 ICSSDKRIACEEEFSDSEEEGEGGRKNSSNFKKAKRVKTEDEKEKDPEEKKEVTEEEKTK 466 MmHDAC1 ICSSDKRIACEEEFSDSDEEGEGGRKNSSNFKKAKRVKTEDEKEKDPEEKKEVTEEEKTK 466

PfHDAC1 VPY------449 TgHDAC3 VPI------451 CpRpd3 ------HsHDAC1 EEKPEAKGVKEEVKLA 482 MmHDAC1 EEKPEAKGVKEEVKLA 482

Figure 5.3 Multiple sequence alignment of class I HDAC amino acid sequences. (A) Schematic illustration of P. falciparum HDAC1 and Human HDAC1 protein structure. The position of the deacetylase core domains are indicated by red and black stripe shading. (B) Alignment of class I HDAC homologues from P. falciparum (PfHDAC1; CAD51938), T. gondii (TgHDAC3; AYY53803), C. parvum (CpRpd3; XP_62348), human (HsHDAC1; CAG46518), and mouse (MmHDAC1; AAI08372). The alignment is between amino acids 1- 499 corresponding to the full length PfHDAC1 with deacetylase core domain is shown in black bars. The acetoin utilization/acetylpolyamine aminohydrolase signature motifs I-VII are shown in red arrows. The two amino acids insertion at the position 95-96 relative to PfHDAC1 is shown in green. Catalytically important histidine residues (H28, H68, H140, H141, H178 and H179) and aspartic acid residue (D174) identified in the mouse HDAC1 (Taplick et al., 2001) and human HDAC1 (Hassig et al., 1998) are marked with asterisks. 79

Chapter 5

5.3.3 In silico PfHDAC1 homology model

A PfHDAC1 homology model was generated to predict the similarity of PfHDAC1 to human

HDAC enzymes and to predict theoretical binding affinity of HDAC inhibitors tested in Chapter

3. This model was constructed using the available crystal structure of the bacterial HDLP and human HDAC8 which have 27% and 37% amino acid sequence identity to PfHDAC1, respectively (Figure 5.4). The residues involved in coordinating the catalytic zinc atom (Asp

174, His 176, and Asp262 of PfHDAC1) and other catalytic important residues (His138,

His139, Asp172, and Tyr301 of PfHDAC1) of PfHDAC1 are common to all three HDAC proteins (Figure 5.4). The hydrophobic residues (Gly147, Phe148, His176, Phe203, and Tyr301 of PfHDAC1) of the active site wall beginning at the entrance to the catalytic zinc atoms are also well conserved in all three HDACs with exception of a conservative substitution from

Leu269 in PfHDAC1 to Methionine in human HDAC8. The active site of PfHDAC1 consists of seven loop regions (Loops 1 – 7). The upper part of the binding site is made up of Loops 1 and

2 and Loops 3 – 7 form the lower region of the binding site that houses the catalytic zinc atom.

Loop 1 of PfHDAC1 is similar to the HDLP as human HDAC8 has two amino insertions (Pro22 and Cys28) in this region. PfHDAC1 and HDLP also contain His21in loop 1 that is absent in human HDAC8. Loop 2 of PfHDAC1 has two residues insertion at position 95 and 96 (Ala95 and Tyr96) which is one residue and two residues longer than HDLP and human HDAC8, respectively. Loop 5 of PfHDAC1 is also shorter than HDLP and human HDAC8 (two and one insertion, respectively). The homology model of PfHDAC1 indicates that this enzyme has a relatively open binding site and appears to have at least three binding pockets at the surface entrance to the zinc-bound groove (Figure 5.4). L-CYS and 2-ASA compounds (discussed in

Chapter 3) flexibly docked into the active site of the PfHDAC1 homology model predicted binding affinities of 1µM – 1nM for these compounds.

Chapter 5

Figure 5.4 Homology model of PfHDAC1. Shown is the hydrophobicity surface (red is hydrophobic, and blue is hydrophilic amino acid residues) with docked inhibitors 2-ASA-8 (orange, carbon atoms) and 2-ASA-13 (green, carbon atoms; blue, nitrogen atoms; red, oxygen atoms), demonstrating three potential binding pockets at the entrance to the active site tunnel containing the catalytic zinc atom (purple).

5.3.4 Analysis of P. falciparum classII HDACs

The P. falciparum genome also contains two homologous to class II HDAC homologues

(Plasmodium genome resource gene ID: PF14_0690 and PF10_0078). Homologues of class II

HDACs are also found in other Plasmodium species. In contrast to PfHDAC1, these

Plasmodium class II HDACs share very limited amino acid sequence identity to each other

(~6% amino acid identity across the full length) and to other eukaryotic homologues (~6% animo acid sequence identity across the full length sequences; Figure S2). The deacetylase domain regions of these homologues contain the deacetylase signature motifs found in members of acetoin utilisation proteins and acetylpolyamine amidohydrolases (Leipe & Landsman,

1997). However, partial consensus in the deacetylase region was observed indicating that the deacetylase catalytic core of these homologues may have diverged from other eukaryotic class

II HDACs (Figure S2). Because of this very limited sequence similarity, in silico modeling studies were not able to be carried out.

Chapter 5

5.3.5 Stage specific expression of PfHDAC1 during the P. falciparum intra-

erythrocytic developmental cycle

The transcription profile of Pfhdac1 was assessed over the 48 hour asexual part of the P. falciparum lifecycle at ~6 hour intervals (Figure 5.5). Four Pfhdac1-specific PCR probes corresponding to the full length Pfhdac1 (nucleotides 1-1350), the N-terminal region

(nucleotides 1-850), the deacetylase domain (nucleotides 15-957), and the C-terminal region

(nucleotides 958-1350) were used in Northern blot analysis (Figure 5.5). The full length

Pfhdac1 and the C-terminal probes detected three transcripts, a doublet at ~4kb and a transcript at ~2kb throughout all stages of the asexual developmental cycle (Figure 5.5). The ~2kb transcript is likely to be due to cross reactivity with another P. falciparum gene as this species was not present using the N-terminal and deacetylase domain probes. Blast searches of the C- terminal of Pfhdac1 against the P. falciparum genome indicate there are 16 hits (transcripts) with greater than 50% nucleotide identity to this region (genes are listed in Table S4). The

Pfhdac1 transcript in trophozoite- and schizont-stages appears as a doublet indicating there may be processing of this gene at the transcriptional level (Figure 5.5). In this study, low expression of Pfhdac1 transcript was also detected in the ring- and early-trophozoite-stages (Figure 5.5) indicating this gene may be transcribed earlier on in the asexual developmental cycle then previous observed (Joshi et al., 1999). Overall, this stage-specific analysis indicates that the

Pfhdac1 is transcribed throughout the asexual developmental cycle of the P. falciparum parasite. The merozoite surface protein 1 (Pfmsp-1) gene was used as a stage-specific control probe and as expected peak expression of this transcript was in the mature schizont stages

(Bozdech et al., 2003) (Figure 5.5).

Chapter 5

2 612182430364041424448 h post invasion 4kb- Full length Pfhdac1 AB 2kb-

4kb- N-terminusPfhdac1 A C 2kb-

4kb- Deacetylasedomain D E 2kb-

4kb- C-terminusPfhdac1 2kb- FB

Pfmsp-1 9kb-

EtBr

Figure 5.5. Stage-specific transcription profile of pfhdac1 during the asexual developmental cycle of P. falciparum. Total RNA was collected from highly synchronous 3D7 P. falciparum parasites starting at ring-stage and then at ~6 hour intervals up to 48 hours. RNA was separated via 0.8% agarose gel electrophoresis, transferred to nitrocellulose membrane and then hybridized with four Pfhdac1 PCR probes corresponding to the full length Pfhdac1 (Primers A and B, nucleotides 1 – 1350), the N-terminal region (Primers A and C. nucleotides 1 – 850), the deacetylase domain (Primers D and E, nucleotides 15 – 957) and the C-terminal region of Pfhdac1 (Primers F and B, nucleotides 958 -1350). Primer sequences are listed in Table S3. The merozoite surface protein 1 (Pfmsp-1) was included as stage specific control. Ethidium bromide (EtBr) stained agarose gel is shown for RNA loading. Molecular size in kilobase (Kb) pairs is shown. Giemsa-stained infected erythrocytes corresponding to ring-, trophozoite- and schizont-stage are shown.

The protein expression of PfHDAC1 at ~6 hour intervals of the 48 h asexual developmental cycle was examined by Western blot. PfHDAC1 antisera was generated against a C-terminal peptide of PfHDAC1, (residue 428 – 445, RRKNYDDDFFDLSDRDQS) as used previously

Chapter 5

(Joshi et al., 1999). The mouse polyclonal anti-sera recognized a band of protein predicted size

~51.3kDa in total protein extracts from mixed asexual-stage P. falciparum parasites (Section

5.2.4). In time course expression studies the anti-PfHDAC1 antisera recognized a band of expected size (~50kDa) beginning at trophozoite-stage (~20 h post invasion) and progressing into the mid to late-schizont-stages (~36 h post invasion; Figure 5.6). As expected, the PfAMA-

1 antisera reacted with a protein of ~44kDa (Treeck et al., 2009) in ring-, late-trophozoite and schizont-stages but not early-trophozoite-stages (Figure 5.6). The anti-PfHSP70 antibody recognized a protein in all parasite protein samples, as expected for this constitutively expressed protein.

26 12 16 24 3036 40 41 42 44 48 h post invasion kDa

55- anti-PfHDAC1 43-

55- anti-PfAMA-1 43-

70- anti-PfHSP70

55-

Commassie staining 70-

Figure 5.6 Stage-specific protein expression of PfHDAC1 during the asexual developmental cycle of P. falciparum. Total protein extracted from highly synchronous 3D7 P. falciparum parasites starting at ring-stage and then at up to 6 hour intervals of 48 hours asexual cycle. Proteins were separated via 12% SDS-PAGE gel electrophoresis, transferred to PVDF membrane and then sequentially stripped and reprobed with anti-PfHDAC1 polyclonal antisera, anti-PfAMA-1 monoclonal antibody as stage specific control or anti-PfHSP70 polyclonal antisera as loading control. Commassie staining showed protein loading. Giemsa-stained infected erythrocytes corresponding to ring-, trophozoite- and schizont-stage are shown.

Chapter 5

As the peak expression of PfHDAC1 was detected in late-trophozoites/schizonts, this stage of the parasites was used to test for deacetylase activity within parasites. As shown in Figure 5.7, deacetylase activity was present in nuclear extracts from trophozoite extracts for two different

P. falciparum lines, Dd2 and D10. To test specificity, deacetylase activity was shown to be inhibited by the HDAC inhibitor trichostatin A at 1µM (Figure 5.7). Similar results were obtained for control HeLa cell nuclear extracts. Together, these expression results suggest that

PfHDAC1 expression is developmentally regulated at the protein level during the asexual developmental cycle in the trophozoite stages.

30000

25000

20000

15000 AFU

10000

5000

0 HeLa Dd2 D10

Figure 5.7 Deacetylase activity in P. falciparum trophozoite nuclear extracts. Nuclear extracts were prepared from trophozoite stage (Dd2 and D10) parasites using the NucBuster protein extraction kit (Calbiochem, USA). ~1 × 108 parasites were used in each reaction. Untreated extract reactions are shown in black bars and activity in extracts treated with 1µM TSA are shown in white bars. HeLa cell nuclear extract (2µg, Upstate, USA) was used in each reaction as positive control. Deacetylase activities are presented as arbitrary fluorescence units (AFU). The mean activities for three independent extracts, each assay was carried out in duplicate are shown (± standard deviations).

5.3.6 Recombinant expression of the PfHDAC1 in E. coli

The ability to extract native deacetylases from in vitro cultures of P. falciparum parasites represents a useful tool for the development of inhibitors to target P. falciparum HDACs. It allows new compounds to be assayed against total deacetylase activity, providing some indication of target-based activity. Unfortunately, these extracts are mixtures of all nuclear proteins and limited to small scale studies. To try to overcome this limitation, attempts were

Chapter 5 made to express functional recombinant PfHDAC1 in E. coli cells. The advantages of using an

E. coli protein expression system is that the process is faster and less expensive than using eukaryotic system based on mammalian or insect cells. However, a disadvantage of using a prokaryotic system to express eukaryotic proteins is that the AT-rich genome (up to 80% for some genes) of P. falciparum can significantly influence protein yield. The A-T content of

PfHDAC1 sequence is 73%. To overcome this issue the pRIG plasmid (Baca & Hol, 2000) which encodes for less represented tRNAs in E. coli (encoding Arg, Ile, and Gly) was co- transformed with the pRSET expression plasmid containing PfHDAC1 (Figure 5.8). This plasmid has been found to improve expression of P. falciparum proteins including aldolase

(Baca & Hol, 2000) and dihydroororate dehydrogenase (DHODH) (Baldwin et al., 2002)

pRSET-PfHDAC1 pRIG-plasmid 4212 bp 5348 bp

Figure 5.8 Schematic representation of the pRSET-PfHDAC1 prokaryotic expression plasmid (A) and pRIG plasmid (B). The full length PfHDAC1 (amino acids 1 – 449, minus the stop codon) was cloned in frame with the N-terminal hexa-histidine (6×His) tag. Expression of recombinant PfHDAC1 is under control of the bacterial T7 promoter. An enterokinase cleavage site (EK) allows for optional removal of hexa-histidine tag N-terminus of PfHDAC1. Plasmid expression is maintained by the Ampicillin selection marker. The pRIG plasmid encoding for tRNAs, ArgU (arginine), ilex (isoleucine), and glyT (glycine).

The pRSET-PfHDAC1 expression vector was co-transformed with the pRIG plasmid (Baca &

Hol, 2000) into BL21 E. coli cells. Following culture and induction with IPTG (Section 5.2.5), a predominant band of ~55kDa corresponding to 6×His-PfHDAC1 protein was obtained in crude

E. coli protein lysates, as shown in by Western blot using anti-PfHDAC1 and anti-RGS-His 86

Chapter 5 antibodies (Figure 5.9; Lane 2, arrows). A protein of ~55kDa was also able to be seen in the corresponding commassie-stained SDS-PAGE gel (Lane 2, arrows; Figure 5.9), but not in control E. coli cell extracts containing either no vector (Lane 1; Figure 5.9) or E. coli cells transformed with the positive control plasmid pRSET-PfDHODH and pRIG (Lane 3; Figure

5.9). The PfHDAC1-6×His protein was expressed in the absence of IPTG induction (Lane 2, uninduced; Figure 5.9) suggesting leaky expression using this expression vector. The control

PfDHODH protein was also overexpressed without IPTG induction (Lane 3, uninduced; Figure

5.9). For deacetylase activity, crude soluble protein extracts from the 6 h inductions were tested.

No activity was detected in any samples (Figure 5.9B). The positive control HeLa cells nuclear extracts (2µg) displayed deaecetylase activity (Figure 5.9B). These results suggest that either the PfHDAC1-6×His concentration was too low in these samples or that it was inactive.

To determine whether any deacetylase activity could be detected in purified material, a small scale (30mL culture) expression and purification was carried out using the same pRSET-

PfHDAC1 E. coli clone. Following affinity purification (Section 5.2.5), a band of ~55kDa corresponding to the expected size of PfHDAC1-6×His was detected in elution fractions 1 and 2 using anti-PfHDAC1 and anti-RGS-His antibodies (Figure 5.10A). The eluted material (elution

2) was tested for deacetylase activity. No activity was detected in this purified material (1µg and

2.5µg in 5µl and 15µL, respectively from 200µL original volume; Figure 5.10B). The positive control HeLa cells nuclear extract (2µg) gave the expected result (Figure 5.10B).

Chapter 5

1mM IPTG 1mM IPTG uninduced 3 h 6 h uninduced 3 h 6 h A C 123123 1 23 1 23 1 23 1 23 kDa kDa

55- 55- 43- 43- 34- 34-

anti-RGS-His anti-PfHDAC1 B D

20000 15000 10000 AFU 55- 5000 43- 0 34-

Commassie

Figure 5.9 Pilot expression of recombinant PfHDAC1 (rPfHDAC1-6×His) in BL21 E. coli cells. Total protein extract in 1× SDS-PAGE sample buffer was separated on a 12% SDS-PAGE gel and transferred to PVDF membrane. (1) BL21 cells with no vector, (2) BL21 cells co- transformed with pRSET-PfHDAC1 and pRIG plasmid or (3) positive control, BL21 cells co- transformed with pRSET-PfDHODH and pRIG plasmid. Cultures were uninduced or induced with 1mM IPTG for 3 h or 6 h. (A) Membranes were probed with anti-RGS-His (1:2000; Qiagen, USA) then stripped and reprobed with (C) anti-PfHDAC1 antisera (1:1000). (B) Commassie stained samples show total protein loading. Arrows indicate the overexpressed PfHDAC1 protein. (D) 15µg of soluble protein extract at 6 h post induction from BL21 no vector, BL21-pRSET-DHODH and BL21-pRSET-PfHDAC1 were examined for deacetylase activity in a fluorometric deacetylase assay (Section 2.10; Upstate, USA). HeLa cell nuclear extract (2µg) supplied with the kit (Upstate, USA) was included as positive control. Deacetylase activity measured as arbitrary fluorescent units (AFU) in single reaction assay is shown.

Chapter 5

Elution Elution Elution B C Wash 3 Wash 3 Wash 3 Unbound Unbound Unbound A Start E1 E2 E3 Start E1 E2 E3 Start E1 E2 E3

kDa kDa kDa

55- 55- 55- 43- 43- 43- 34- 34- 34-

Commassie anti-RGS-His anti-PfHDAC1

D 1000

750

500

AFU

250

0 HeLa 1µg 2.5µg rPfHDAC1-6 His

Figure 5.10 Small scale expression and purification of rPfHDAC1-6×His. A 30mL culture of E. coli containing the pRSET-PfHDAC1 and pRIG plasmid was grown in LB broth and induced with 1mM IPTG for 6 h. Cells were pelleted and soluble fractions were collected and subjected to purification (Section 5.2.5). (A) Protein fractions were resuspended in 1× SDS- PAGE sample buffer then resolved via a 12% SDS-PAGE gel electrophoresis and transferred to PVDF membrane. Samples shown are starting material prior to commencing purification (0.05 volume of 300µL; starting material), flow through material after overnight incubation with cobalt affinity resin (0.05 volume of 300µL; unbound material), wash 3 (0.005 volumes of 3000µL), Elution E1 (0.05 volume of 300µL), E2 (0.05 volume of 300µL) and E3 (0.05 volume of 300µL). Protein fractions were probed with anti-RGS-His antibody (1:2000) and anti- PfHDAC1 antibody (1:1000). Commassie stained samples was included to show protein loading. (B) Deacetylase activity was examined for rPfHDAC1 E2 (1µg and 2.5µg). HeLa cell nuclear extract (2µg) supplied with the kit (Upstate, USA) was included as positive control. Deacetylase activity measured as arbitrary fluorescent units (AFU) in single reaction assay is shown.

To determine if cleavage to remove the N-terminal 6×His tag using enterokinase could improve protein activty, a large scale expression and purification processes was carried out. Following purification, a band of expected size (~55kDa) was detectable by commassie staining in the first elution fraction (Figure 5.11A). However, when Western blot was carried out using anti-

PfHDAC1 anti-sera, the 55kDa band was not present (Figure 5.11A). Instead, a small band of

~35kDa was detected (but not in the starting material), indicating that the protein had been degraded. While protease inhibitor cocktail (Roche, Germany) was included in all the buffers

Chapter 5 used in the purification process and purification was carried out at 40C, clearly this did not inhibit all protease activity. As might be expected the eluted protein showed no deacetylase activity (Figure 5.11B). To ensure that the concentrated elutate did not contain inhibitory material, HeLa cell nuclear extract was spiked with 15µg (in 15µL) but this did not affect deacetylase activity in this control extract (Figure 5.11B).

Wash Elution A Start Unbound 1357 9 123Pool kDa

B 600 55- anti-PfHDAC1 500

43- 400

34- 300 AFU 200

100

0 55- HeLa Hela + rPfHDAC1 rPfHDAC1 43- Commassie 34-

Figure 5.11 Large scale expression and purification of rPfHDAC1-6×His. An E. coli clone containing the pRSET-PfHDAC1 and pRIG plasmids was grown in LB broth (1 Litre) and induced with 1mM IPTG for 6 h. Cells were pelleted and soluble fractions were collected and subjected to purification (Section 5.2.5). (A) Protein fractions were resuspended in 1× SDS- PAGE sample buffer then resolved via a 12% SDS-PAGE gel electrophoresis and transferred to PVDF membrane. Samples shown are starting material prior to commencing purification (0.0015 volume of 10mL; starting material), flow through material after overnight incubation with cobalt affinity resin (0.0015 volume of 10mL; unbound material), wash 1, 3, 5, 7, and 9 (0.0015 of 10mL), Elution 1, 2, 3 and a sample comprised pooled and concentrated elution fractions (pool), (0.015 volume of 1mL). Commassie stained samples were included. (B) Deacetylase activity of purified rPfHDAC1 fraction (15µg in 15µL) was examined. HeLa cells (2µg) nuclear extract supplied with the kit (Upstate, USA) was included as positive control. The same of amount of HeLa nuclear extract spiked with 15µg of purified rPfHDAC1 fraction was included to test if fraction has any inhibitory factors. Deacetylase activity measured as arbitrary fluorescent units (AFU) in single reaction assay is shown.

5.4 Discussion

The identification of PfHDAC1 a drug target in malaria parasites is based on the ability of eukaryotic class I/II HDAC inhibitors to kill P. falciparum parasites and to cause histone hyperacetylation. Apart from these data, relative little was known about this protein in

Plasmodium. To learn more about PfHDAC1 a detailed phylogenetic analysis was first carried out. These studies show that all Plasmodium parasites, for which genome sequence data is 90

Chapter 5 available, possess a class I HDAC homologue. The Plasmodium class I HDAC homologues are very closely related and share >90% amino acid identity (Figure S1). Phylogenetically, the closet relatives of PfHDAC1 and its Plasmodium homologues are from the apicomplexan parasites, Toxoplasma gondii and Crytosporidium. The deacetylase catalytic domain (~319 aa) located at the N-terminus of PfHDAC1 shares ~65% identity with human and mouse HDAC1 at the amino acid level.

Compared to higher eukaryotic proteins, a noticeable difference in apicomplexan parasites class

I HDACs is a two amino acid insertion at position 95 and 96, relative to PfHDAC1.

Interestingly, mutagenesis studies of the T. gondii class I HDAC (TgHDAC3) has shown that these insertions may be partially associated with resistance to a cyclic peptide HDAC inhibitor

(FR235222) (Bougdour et al., 2009). In contrast, nothing is known about if or how HDAC inhibitor resistance might be achieved in P. falciparum. Therefore, similar mutagenesis studies in the future will be useful in elucidating possible mechanism of resistance to HDAC inhibitors in P. falciparum. This information might also help guide the design and development of next generation of HDAC inhibitors.

The N-terminal region of class I HDACs also contains sequence motifs important for protein interactions. For example, the N-terminal amino acid residues 1-51 of the mouse HDAC1 have been shown to facilitate homodimerisation, and hetero-dimerisation with HDAC2 and HDAC3

(Taplick et al., 2001). The N-terminal amino acid stretch of PfHDAC1 is ~50% identical to the mouse sequence. Although it is tempting to speculate that this region of PfHDAC1 may also facilitate protein-protein interactions, there is currently no evidence for this. Data to suggest multiprotein complexes exist in T. gondii are however available (Saksouk et al., 2005). Future studies to test this in P. falciparum might include immunoprecipitation (IP) studies or transgenic approaches such as tandem affinity purification (TAP-tag) (Takebe et al., 2007).

Chapter 5

The C-terminal region of higher eukaryotic class I HDACs are crucial for nuclear localization and contain a classical nuclear localization signal (NLS) consisting of a short lysine-rich sequence (Taplick et al., 2001) (human and mouse HDAC1 starting at residue 438

KKAKRVK). This sequence, when fused to the report protein green fluorescent protein (GFP), has been shown to target GFP to the cell nucleus (Taplick et al., 2001). A search for this NLS in

PfHDAC1 using the nuclear signal prediction tool PredictNLS (Cokol et al., 2000) revealed no similar NLS in PfHDAC1. Interestingly, previous studies have shown the putative P. falciparum class II HDAC (PlasmoDB genome resource gene ID: PF14_0690 (PfHDAC2)) and a putative

RNA polymerase (PF14_0207), both possess lysine-rich NLS sequences identified using the

PredictNLS tool (Cokol et al., 2000). These putative NLS sequence when fused to the GFP reporter protein, were able to translocate GFP into the cell nucleus of CHO-K1 cells (Chan et al., 2006) indicating that some Plasmodium proteins may utilise similar nuclear targeting mechanism to mammalian cells. Using an alternative NLS prediction search tool (cNLS

Mapper) (Kosugi et al., 2009) a bipartite NLS sequence at the N-terminal end of PfHDAC1 was

(weakly) predicted, residue 3 – 31 (NRKKVAYFHIGSYYYGAGHPMKPQRI). A classical bipartite NLS is generally made of KRKX10-12K(K/R)(K/R), where X is any amino acid residue of length between 10 – 12 residues (Robbins et al., 1991). A recent study showed that a linker length up to 20 residues could also function as NLS (Kosugi et al., 2009), indicating the predicted bipartite NLS of PfHDAC1 may function as its NLS. A possible approach to test this putative NLS sequence might be to express the predicted PfHDAC1 NLS as a fusion reporter protein in P. falciparum and then examine the subcellular localization of this protein.

A major consideration in the development of any new drug is to limit host toxicity and this is particularly important when the target is present in both pathogen and host. To begin to understand how similar the PfHDAC1 is to human HDACs, an in silico homology model was generated. The homology model of PfHDAC1 is based on the crystal structure of the HDLP and human HDAC8 and indicates these enzymes share a structurally similar zinc dependent active site. Catalytically important residues are highly conserved in all three of these HDACs (Figure

Chapter 5

5.3; His138, His139, Asp172 and Tyr301 of PfHDAC1). However, PfHDAC1 appears to be different at the entrance to the active site which has a relatively open binding site and at least three binding pockets (Figure 5.3). This suggests that there are differences that may be able to be exploited to synthesise parasite-selective compounds. A recent homology model study of

PfHDAC1 (Mukherjee et al., 2008) also described similar results to this study. Furthermore, ligand docking studies using the PfHDAC1 model demonstrated good correlation between the biological activity of hydroxamate-based HDAC inhibitors (TSA, ABHA, SBHA, and SHA) and the interaction profile of the docked posed of these molecules in PfHDAC1 (Mukherjee et al., 2008). These findings indicate that PfHDAC1 homology model is a useful tool for pre- screening new compounds.

Information on when PfHDAC1 is expressed in the parasite may be useful to better target the

PfHDAC1 protein using inhibitors. To begin to understand how PfHDAC1 might be regulated in the intra-erythrocytic developmental cycle, a detailed expression profile study was carried out. Data from these experiments demonstrate that Pfhdac1 is transcribed throughout the intra- erythrocytic developmental cycle (Figure 5.5). Interestingly, a doublet transcript of Pfhdac1 was observed in trophozoite and schizont parasites but not in ring and early trophozoites indicating there might be an alternative transcription start site for Pfhdac1. Protein expression profiling of

PfHDAC1 protein indicates it might be developmentally expressed in the trophozoite- and schizont-stages (Figure 5.6). Using this expression information, assays were next carried out to demonstrate that nuclear extracts prepared from trophozoites contain deacetylase activity and that this activity can be inhibited by the HDAC inhibitor TSA (Figure 5.7). As detailed in this

Chapter, this expression and activity information has been useful for mode of action studies.

While crude extracts do provide a useful tool, clearly recombinant PfHDAC1 would allow more detailed mode of action studies and higher through-put assay to be carried out. Therefore, recombinant PfHDAC1 was prepared in E. coli cells in order try to generate enough functional protein for enzymatic studies. While this protein was shown to be expressed in a soluble form

Chapter 5 that could be purified, it was inactive. The reason behind this inactivity was not fully explored due to time constraints. However, expression of eukaryotic proteins in E. coli often results in incorrect folding, aggregation, and inclusion body formation, as prokaryotic expression systems may be lacking in natural binding partners or posttranslational processing machinery which are required for proper folding. While, PfHDAC1 may also be posttranslationally processed this remains to be determined. Although active recombinant PfHDAC1 was not obtained from E. coli during the current study, it has very recently been obtained using synthetic codon optimized gene and expression in insect cells (Patel et al., 2009). This enzyme is active in deacetylase assays and is inhibited by HDAC inhibitors. The PfHDAC1 protein has kinetic constants (Kcat,1)

-1 0.19 ± 0.01 s and (Km,1) 30 ± 2µM for a peptide substrate (Patel et al., 2009). This recombinant

PfHDAC1 was also successfully used to test a panel of hydroxamate HDAC inhibitors with levels of activity (IC50 20 – 110nM) correlating well with the kinds of nuclear extract activity obtained for compounds in this study (Chapter 4) and by others using a similar approach to that described here (Darkin-Rattray et al., 1996, Dow et al., 2008). The expression vector containing codon optimized PfHDAC1 has been obtained and while beyond the scope of this thesis, will be useful in validating the activity of compounds in this study and other function studies including determination of substrate specificity, enzyme kinetics and crystallography.

Chapter 6

Generation and characterisation of PfHDAC1 “disruption” and

“overexpression” mutants

Chapter 6

6.1 Introduction

PfHDAC1 is likely to be essential to the intra-erythrocytic development of P. falciparum and thus a good antimalarial drug target. However, the hypothesis that this enzyme is essential needs to be confirmed. In this chapter, transgenic parasites were generated to experimentally examine the function and essential nature of PfHDAC1. Transgenic parasite lines overexpressing full length PfHDAC1 were also generated to confirm the nuclear localisation of PfHDAC1 and to determine if overexpressed PfHDAC1 can alter the susceptibility of P. falciparum parasites to different HDAC inhibitors. This latter approach has been successfully used in previous studies that have examined the target of P. falciparum M17 leucyl (Gardiner et al., 2006) and M18 aminopeptidases (Teuscher et al., 2007) inhibitors. In these studies overexpression mutants of the two aminopeptidases were shown to have a reduced sensitivity profile to selected inhibitors.

6.2 Materials and methods

6.2.1 Generation of gene disruption constructs

To generate a Pfhdac1 (PFI1260c) disruption plasmid, a 852 base pair (bp) Pfhdac1 fragment, beginning at the start codon was PCR amplified from 3D7 gDNA using standard PCR conditions (Section 2.16) with primers: HDAC1-F-ArvII 5’

TTcctaggATGTCTAATAGAAAAAGG 3’ and HDAC1-R-ClaI

5’GGatcgatTTCTACACATCTGGCATGACC 3’. The resulting PCR fragment, referred to as

Pfhdac1-KO, was cloned into the pGEM-T vector (Section 2.18; Promega, USA). Sequencing was carried out to verify the pGEM-T vector had the correct Pfhdac1 insert (Section 2.17). The fragment was digested with restriction enzymes ArvII and ClaI (Section 2.20; New England

Biolabs, UK) according to manufacturer’s instructions. The Pfhdac1-KO insert was directionally sub-cloned into the pCHH vector containing the human dihydrofolate reductase

(hDHFR) cassette to confer resistance to the antifolate WR99210. The expression of the hDHFR in this vector is under the control of the P. falciparum camodulin (Cam 5’) promoter.

The final knockout vector, referred to as pCHH-Pfhdac1-KO, was transfected into 3D7 P. falciparum infected erythrocytes using the following electroporation conditions; 2.5kV, 200Ω,

Chapter 6 and 25µF (Section 2.24). Parasites resistant to WR99210 were observed on Giemsa-stained thin blood smears 21 – 30 days post transfection. Three cycles of WR99210 drug selection were then carried out to force integration. Parasites were cultured in the presence of 10nM WR99210 for three weeks, then culture with no drug for two weeks. Genomic DNA was then extracted from the pCHH-Pfhdac1-KO parasite population (Section 2.15) and integration of the pCHH-

Pfhdac1-KO plasmid into the parasite genome was determined using standard PCR conditions

(Section 2.16). Primers used are listed in Table S3.

6.2.2 Generation of overexpression constructs

The full length Pfhdac1 (PFI1260c) gene, minus the stop codon, was amplified from 3D7 P. falciparum genomic DNA using standard PCR conditions (Section 2.16) with primers: HDAC1-

F-BglII 5’ GGctgtctATGTCTAATAGAAAAAAGG 3’ and HDAC1-R-PstI 5’

AActgcagATATGGTACAATAGATTGATCC 3’. The amplified PCR products, were cloned into the pGEM-T vector (Promega, USA). Individual plasmid clones were selected, plasmid

DNA prepared (Section 2.19), and DNA sequenced (Section 2.17) to confirm the correct insert was present. The full length Pfhdac1 product was then digested with restriction enzymes BglII and PstI and ligated in frame with the cmyc epitope tag (5’GCCTGAACAA AAACTCATAA

GCGAAGAAGA TTTATAAATG CA3’) or green fluorescent protein (GFP) that had been previously cloned into P. falciparum-modified pENTR™-cmyc and pENTR™-GFP plasmids

(Gateway, Invitrogen, USA, a gift from Dr Donald Gardiner, QIMR). These plasmids contain the kanamycin resistance marker for plasmid maintenance in E. coli cells. The entry vectors

(pENTR-PFI1260c-cmyc and pENTR-PFI1260c-GFP) were chemically transformed (Section

2.23) into TOP10 E. coli cells and plated onto Luria Broth (LB) agar plates containing 50µg/mL

Kanamycin (Sigma, USA). Mini-prep plasmid DNA was prepared and clones screened for the presence of correct insert by restriction digestion with the enzymes BglII and PstI and DNA

® sequencing. The entry vectors were subjected to LR Clonase reactions to recombine the insert with a NcoI-linearised P. falciparum- modified Destination vector containing the hDHFR cassette conferring resistance to the antifolate WR99210, according to manufacturer’s

Chapter 6 instructions (pDEST®, Invitrogen, USA, a gift from Dr Donald Gardiner, QIMR). The final transfection vectors, referred to as pHH1-PFI1260c-cmyc.TV and pHH1-PFI1260c-GFP.TV were chemically transformed (Section 2.23) into TOP10 E. coli cells and grown in LB agar plates containing 100µg/mL ampicillin (Sigma, USA). Mini-prep plasmid DNA was prepared and restriction digests and DNA sequencing carried out to confirm the final clones contained the correct inserts. Midi-prep plasmid DNA was prepared using a Midi-prep plasmid extraction kit, according to the manufacturer’s instructions (Qiagen, USA). The final plasmid constructs (50 -

100µg/transfection) were transfected into P. falciparum D10 parasites using the following electroporation conditions, 2.5kV, 200Ω, and 25µF (Section 2.24). P. falciparum transfectants were grown under normal culture conditions (Section 2.1) in the presence of 5nM WR99210.

Parasites positively selected using WR99210 were observed on Giemsa-stain thin blood smears

21 – 30 days post transfection. Cultures were then maintained on 10 nM WR99210 for episomal expression of PfHDAC1-cmyc and PfHDAC1-GFP. In some cases WR99210 levels were increased to50 nM WR99210 for drug susceptibility and enzymatic assay studies.

6.3 Results

6.3.1 Characterisation of disruption mutants

To investigate whether PfHDAC1 is essential to parasite development, as would be predicted of a good antimalarial drug target, a single homologous crossover recombination strategy was employed to attempt to genetically disrupt Pfhdac1. A gene disruption construct was generated containing a 852bp fragment of Pfhdac1, as described in Section 6.2.1 (Figure 6.1). Parasite populations positive for the pCHH-Pfhdac1-KO plasmid were subjected to “positive/negative” drug cycling with WR99210 (Section 6.2.1) to force integration of the plasmid into the Pfhdac1 locus.

Chapter 6

ClaI

ArvII

a pCHH-Pfhdac1-KO 7449 bp

Figure 6.1 Schematic representation of pCHH-Pfhdac1-KO knockout plasmid. The plasmid contains a 852bp fragment of Pfhdac1, beginning at the start codon of Pfhdac1 and the human dihydrofolate reductase (hHDFR) cassette conferring resistance to the antifolate WR99210 under the control of the P. falciparum calmodulin (CAM 5’) promoter and (HRP2 3’) stop cassette.

PCR analysis of genomic DNA prepared from these parasites was carried out to determine whether the vector had integrated into the correct genomic locus. The predicted integration scenario of a single copy of the plasmid is shown in Figure 6.2A. The Pfhdac1-KO fragment should integrate into the Pfhdac1 locus following drug cycling. Control PCR reactions to amplify genomic Pfhdac1 were positive in both 3D7 wildtype and transfected lines (Lanes 2 and 10 (full length, 1.35kb), and Lanes 8 and 16 (deacetylase domain, 0.9kb); Figure 6.2B).

Likewise, a control PCR using a transfection plasmid-specific primer combination was only positive for a fragment of the expected size of 1.97kb in the transfectants (Lanes 3 and 11;

Figure 6.2B). Control PCR reactions to test the primer to the 5’ untranslated region of Pfhdac1 were positive in both wiltype and transfected lines (Lanes 4 and 12; Figure 6.2B) with a PCR product of expected size of ~0.9kb obtained. This product is made up of 88bp upstream from the start codon of Pfhdac1 and the 850bp Pfhdac1-KO 5’ fragment. As shown in Figure 6.2B, fragments specific to the pCHH-Pfhdac1-KO vector were amplified from genomic DNA of the transfected line (Lane 11 and 14), but not in wildtype genomic DNA. The PCR product in Lane

11 was amplified with the forward primer specific to Pfhdac1 (primer a; Figure 6.2A) and the

Chapter 6 reverse primer specific to hDHFR gene in the vector (primer f; Figure 6.2A). The PCR product in Lane 14 was amplified with primers specific to the plasmid backbone and should only be positive for the plasmid (primers h and I; Figure 6.2A). Subsequently, the primer to the 5’ untranslated Pfhdac1 region (primer g; Figure 6.2A) was combined with hDHFR reverse primer to test for plasmid integration at the 5’ end of Pfhdac1 locus. This primer set resulted in a negative PCR product in both wildtype and transfected lines (Lane 5 and 13; Figure 6.2B) indicating that integration of the plasmid into the Pfhdac1 locus had not taken place. To confirm this finding the predicted integration event at the 3’ end of Pfhdac1 was examined. A forward primer to the vector backbone (primer h; Figure 6.2A) was combined with the Pfhdac1 reverse primer (primer c; Figure 6.2A). Consistent with the negative 5’ integration result, the expected

PCR product of ~2.85kb (vector backbone of ~1.5kb plus full length Pfhdac1 of 1.35kb) was also negative in both wildtype and transfected lines (Lane 7 and 15; Figure 6.2B). Together, these PCR results suggest that integration of pCHH-Pfhdac1-KO plasmid at the Pfhdac1 was not achieved.

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A Pfhdac1-KO hDHFR pcHH-Pfhdac1-KO HRP2 3’ Cam 5’ UTR

5’ 3’ 3D7 gDNA Pfhdac1

a 5’ 3’

g a b f h i d e c 3D7 wildtype 3D7 transfectants B Marker water a + c a + f g + b g + f h + i h + c d + e a + c a + f g + b g + f h + i h + c d + e 3.0kb- 2.0kb- 1.65kb-

0.85kb-

Figure 6.2 PCR analysis of pCHH-Pfhdac1-KO transfected 3D7 P. falciparum parasites following selection for integrated plasmid. (A) Schematic representation of the predicted single homologous cross-over integration event at the Pfhdac1 locus. Letters represent primers used in PCR analysis and arrows indicate orientation of primers (primer details are listed in Supplementary Table S3). (B) Lane 1, water control; Lanes 2 – 8, 3D7 wildtype gDNA; Lane 9, 1kb Plus ladder (Marker; Invitrogen, USA); and Lanes 10 – 16, gDNA from pCHH-Pfhdac1- KO transfected parasites. The following primer pairs indicate above lane were used: primers a andcC (Lane 2 and 10), primers a and f (Lanes 3 and 11), primers g andbB (Lanes 4 and 12), primers g and f (Lanes 5 and 13), primers h and i (Lanes 6 and 14), primers h and c (lanes 7 and 15), and primers d and e (Lanes 8 and 16). Size of 1kb DNA marker bands are shown in kilobases.

6.3.2 Generation and analysis of P. falciparum PfHDAC1 overexpression mutants

6.3.2.1 Generation of overexpression mutants

In order to overexpress PfHDAC1 in P. falciparum infected erythrocytes, transfection vectors were generated containing full length Pfhdac1, minus the stop codon, fused to a cmyc epitope tag or green fluorescent protein (GFP) (Figure 6.3). Following transfection into ring-stage D10

P. falciparum parasites and positive selection using WR99210, episomal expression of

PfHDAC1-cmyc and PfHDAC1-GFP proteins in these transgenic parasites was maintained using 10nM WR99210 in culture media. WR99210 were increased to 50nM for drug susceptibility and enzymatic assay studies.

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A BglII B BglII

PstI pHH1-PFI1260c-GFP pHH1-PFI1260c-cmyc PstI 8883 bp 8205 bp

Figure 6.3 Schematic representation of PfHDAC1 overexpression plasmids. The full length Pfhdac1 (PFI1260c) was cloned in frame with GFP (A) or the cmyc-epitope (B) in a modified P. falciparum transfection vector (pHH1). These introduced genes are under the control of the Heat-shock protein 86 promoter (HSP86 5’) promoter. The human dihydrofolate reductase (hHDFR) cassette is under the control of the P. falciparum calmodulin promoter (CAM 5’) as a selectable marker, conferring resistance to the antifolate WR99210.

6.3.2.2 Confirmation of expression of PfHDAC1

Northern blots were carried out on RNA prepared from D10 PfHDAC1 overexpression transgenic parasites to confirm the presence of Pfhdac1-cmyc and Pfhdac1-GFP transcript was present. When RNA was probed with a Pfhdac1 specific probe, a transcript of ~4kb corresponding to Pfhdac1 was present in D10 wildtype parasites (Figure 6.4A). However, this

Pfhdac1 transcript was absent in overexpression mutants (Figure 6.4). When RNA was hybridised with 3’ Pbdt or gfp probes, no transcript was detected in wildtype D10, as expected

(Figure 6.4B and C). In D10 parasites transfected with pHH1-PFI1260c-cmyc plasmid, multiple transcripts were detected when hybridised with Pfhdac1 and 3’Pbdt probes and a smeared pattern was seen when RNA was hybridised with gfp probe. A prominent band slightly less than

4kb is consistently present in the cmyc transgenic line (Figure 6.4, white arrows). Multiple transcripts were also detected in the GFP transgenic line, with a prominent band slightly greater than 4kb (Figure 6.4, red arrows) detected using the Pfhdac1, 3’Pbdt, and gfp probes. This corresponds to the Pfhdac1-GFP transcript. A transcript (~2kb) was present in all parasites when probed with a Pfhdac1 or gfp specific probes.

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A BCD R T R TRTTR GFP GFP GFP D10 D10 GFP cmyc cmyc D10 D10 GFP D10 GFP cmyc cmyc D10 D10 cmyc D10 GFP GFP cmyc D10 cmyc cmyc D10

4kb-

2kb-

Pfhdac1 3’Pbdt gfp EtBr

Figure 6.4 Northern blot analysis of PfHDAC1 overexpression mutants. Total RNA from synchronous rings (R) and trophozoites (T) from D10 wildtype (D10), D10 transfected with pHH1-PFI1260c-cmyc plasmid (cmyc), and D10 transfected with pHH1-PFI1260c-GFP plasmid (GFP) were separated via 0.8% agarose gel electrophoresis then RNA transferred to nitrocellulose membrane. The membrane was probed with Pfhdac1 PCR probe, then sequentially stripped and reprobed with a 3’Pbdt probe (excised for pHH1-PFI1260c-cmyc plasmid), and a gfp PCR probe. Black arrows indicate Pfhdac1 transcript, white arrows indicate Pfhdac1-cmyc transcript, and red arrows indicate Pfhdac1-GFP transcript. Ethidium stained (EtBr) RNA was included as loading control. RNA size in kilobases is shown.

Western blots were carried out to examine overexpression at the protein level. Using anti-

PfHDAC1 anti-sera (Section 5.2.3), a protein of the expected size of 51.3kDa was detected in both D10 wildtype and transgenic lines (Figure 6.5). In D10 PfHDAC1-cmyc parasites, a doublet comprising a band the same size as the band in D10 wildtype parasites and a slightly larger band corresponding to the expected mobility of PfHDAC1-cmyc was detected (Figure

6.5, asterisk). The anti-PfHDAC1 anti-sera was not able to recognise the PfHDAC1-GFP protein but a protein of ~77kDa corresponding to PfHDAC1-GFP was detected using anti-GFP anti-sera. The lack of PfHDAC1-GFP signal with the anti-PfHDAC1 anti-sera (which was generated using a C-terminal peptide) may be due to the large GFP tag interfering with the C- terminal end of PfHDAC1. As expected no protein band was detected in D10 or D10

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PfHDAC1-cmyc using anti-GFP antisera. Anti-cmyc antisera was examined but was not able to detect the PfHDAC-cmyc product (not shown).

kDa D10 cmyc GFP 70 –

anti-PfHDAC1 55 – *

70 – anti-GFP 55 –

34 – anti-GAPDH

Figure 6.5 Western blot analysis of PfHDAC1 overexpression mutants. Total P. falciparum protein extracts were separated via 10% SDS-PAGE gel electrophoresis then protein transferred to PVDF membrane and probed with anti-PfHDAC1 anti-sera, anti-GFP anti-sera or anti- GAPDH anti-sera (a kind gift from Dr Daubenberger, Swiss Tropical Institute). Protein extracts are D10 wildtype (D10), D10 transfected with pHH1-PFI1260c-cmyc plasmid (cmyc), and D10 transfected with pHH1-PFI1260c-GFP plasmid. Asterisk indicates PfHDAC1-cmyc co- migrating with endogenous PfHDAC1. Size in kiloDaltons (kDa) is shown. Details of anti-sera are shown in the Table S2.

6.3.2.3 Localisation of PfHDAC1-cmyc and PfHDAC1-GFP fusion protein within

P. falciparum infected erythrocytes

To determine whether the PfHDAC1-cmyc and PfHDAC1-GFP fusion proteins display similar subcellular localisation to endogenous PfHDAC1, immunofluorescent assays were carried out.

Anti-PfHDAC1 anti-sera (Section 5.2.3) localised PfHDAC1 protein predominantly to the parasite nucleus (co-localised with Hoechst DNA staining) in both D10 and transfected lines

(Figure 6.6). The subcellular localisation patterns using anti-PfHDAC1 anti-sera in the transgenic parasites was similar to that obtained for D10 (Figure 6.6B and C) and Dd2 P. falciparum infected erythrocytes (not shown). However, the anti-cmyc and anti-GFP anti-sera failed to detect PfHDAC1-cmyc and PfHDAC1-GFP, respectively (Figure 6.6B and C) in D10 transgenic parasites. Live (unfixed) D10 parasites transfected with the PfHDAC1-GFP plasmid 104

Chapter 6 display fluorescence throughout the parasite cytoplasm and this expression appears to be constitutively expressed in all of the intra-erythrocytic developmental stages (Figure 6.7).

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ABCD10 wildtype D10 PfHDAC1-cmyc D10 PfHDAC1-GFP 5µm Cy2 alone Cy2 Cy2 alone Cy2 anti-mouse anti-mouse anti-mouse anti-mouse anti- HDAC1 anti- HDAC1 Pf Pf anti-cmyc anti-GFP anti-cmyc anti-GFP Bright Field Hoechst Cy2 Merge Bright Field Hoechst Cy2 Merge Bright Field Hoechst Cy2 Merge Figure 6.6 Immunoflourescent assay (IFA) of D10 wildtype and transgenic overexpression parasites. (A) D10 wildtype infected erythrocytes, (B) D10 infected erythrocytes transfected with pHH1-PFI1260c-cmyc plasmid (D10 PfHDAC1-cmyc, maintained on 10nM WR99210) or (C) pHH1-PFI1260c-GFP plasmid (D10 PfHDAC1-GFP, maintained on 10nM WR99210) were fixed in 10% methanol-acetone and hybridised with anti-PfHDAC1 antisera, anti-cmyc antisera (Sigma, USA), or anti-GFP antisera (Roche, Germany) (primary antibodies at 1:500 dilution) then hybridized with goat anti-mouse Cy2 antisera (1:500, Sigma, USA). Infected erythrocytes hybridised with goat anti-mouse Cy2 antisera only were included as negative control. Bright field images show parasitised erythrocytes, Hoechst DNA staining in blue (excitation: 355nm and emission: 460nm); and Cy2 staining in green (excitation: 485nm and emission: 520nm). Images were captured in an AxioSkop 2 Mot Plus microscope (Ziess, USA).

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5µm Early Trophozoite

Mid Trophozoite

Late Trophozoite

Schizont

Bright field Hoechst GFP Merge

Figure 6.7 Live (unfixed) fluorescence images of D10 PfHDAC1-GFP transgenic parasites. Different stages of D10 infected erythrocytes transfected with the pHH1-PFI1260c-GFP plasmid (maintained on 10nM WR99210) were stained with Hoechst and fluorescence detected using a DeltaVision deconvolution microscope (Applied Precision, USA). Bright field images show parasitized erythrocytes, Hoechst DNA staining in blue was detected at wavelength 355nm (excitation) and 460nm (emission), and GFP signal was detected at wavelength 485nm (excitation) and 520nm (emission).

6.3.2.4 Comparison of intra-erythrocytic growth of PfHDAC1 overexpression

mutants versus D10 wildtype parasites

To determine whether overexpression of PfHDAC1 has an effect on parasite intra-erythrocytic development, the growth of the mutants was compared to D10 wildtype parasites via microscopic evaluation of Giemsa-stained thin blood smears. The growth of D10 and transgenic lines was monitored for 90 hours (~2 invasion cycles) starting from ring-stage (Figure 6.8).

These data demonstrate that the growth of PfHDAC1 transgenic parasites (maintained on 10nM

WR99210) was similar to D10 wildtype parasites, with average reinvasion rates (calculated by

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Chapter 6 determining the average parasitemias at 45 h divided by average starting parasitemia) of 4.6 fold, 5 fold, and 6.5 fold for D10, D10 PfHDAC1-cmyc, and D10 PfHDAC1-GFP, respectively.

This re-invasion rate is consistent with other literature reports (D10 wildtype parasite of 4 – 5 fold) (Walliker et al., 1987). Furthermore, differential counts of Giemsa-stained thin blood smears showed that the D10 PfHDAC1 transgenic parasites developed normally, with trophozoites able to develop into schizonts (some residual schizonts observed at ring-stage time points, 45 h) and then producing rings (Figure 6.9).

Assay - 1 16 14 12 10 8 start 24 h 45 h 6 4 2 % TotalParasitemia 0

0 24456290 D10 Hour Post-invasion

D10 c-myc GFP

Assay - 2 cmyc 16 14 12 10 8 GFP 6 4 2 % TotalParasitemia 0 0 24456290

Hour Post‐Invasion

D10 c-myc GFP

Figure 6.8 Comparison of overexpression mutants versus wildtype in vitro growth and development. The growth of synchronous ring-stage parasites (beginning at 1-2% parasitemia) as monitored via microscopic examination of Giemsa-stained thin blood smears is shown for D10 wildtype (D10) compared to D10 PfHDAC1-cmyc (cmyc) and D10 PfHDAC1-GFP (GFP) transgenic parasites (grown in the presence of 10nM WR99210 in culture media). Error bars indicate the standard deviation of three counts on the same blood smear. Giemsa-stained infected erythrocytes show parasite morphology at the start of the experiment and then 24 and 45 h post-invasion.

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D10 - Assay - 1 D10 - Assay - 2 16 16 14 14 12 12 10 10 8 8 6 6 4 4 % Parasitemia % Parasitemia 2 2 0 0 Rings Trophs Schizonts Total Rings Trophs Schizonts Total

0h 24h 45h 62h 90h 0h 24h 45h 62h 90h

cmyc - Assay - 1 cmyc - Assay - 2 16 16 14 14 12 12 10 10 8 8 6 6 4 4 % Parasitemia % Parasitemia 2 2 0 0 Rings Trophs Schizonts Total Rings Trophs Schizonts Total

0h 24h 45h 62h 90h 0h 24h 45h 62h 90h

GFP - Assay - 1 GFP - Assay - 2 16 16 14 14 12 12 10 10 8 8 6 6 4 4 % Parasitemia % Parasitemia 2 2 0 0 Rings Trophs Schizonts Total Rings Trophs Schizonts Total

0h 24h 45h 62h 90h 0h 24h 425h 62h 90h

Figure 6.9 Differential parasitemia counts of overexpression mutants versus wildtype parasites. The growth of synchronous ring-stage parasites (beginning at 1-2% parasitemia) as monitored via microscopic examination of Giemsa-stained thin blood smears (Figure 6.8). Shown are differential counts of D10 wildtype (D10) compared to D10 PfHDAC1-cmyc (cmyc) and D10 PfHDAC1-GFP (GFP) transgenic parasites (grown in the presence of 10nM WR99210 in culture media) monitored over ~90 hour (~2 invasion cycles). Error bars indicate the standard deviation of three counts on the same blood smear.

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6.3.2.5 Susceptibility of PfHDAC1 overexpression mutants to antimalarial

compounds

The susceptibility of D10 PfHDAC1 transgenic parasites to the control drug quinine, and the

HDAC inhibitors SAHA, 2-ASA-9, and 2-ASA-14 was compared to D10 wildtype parasites.

There was no difference in drug sensitivity to quinine for D10 wildtype parasites versus tranfected parasites (IC50 values range between 57 – 67nM, Figure 6.10). Likewise, there was no difference in sensitivity of the mutants versus D10 wildtype parasites to the various hydroxamate-based HDAC inhibitor compounds (Figure 6.10).

QNE SAHA 120 120 100 100 80 80 60 60 40 40 20 20 0 0 -20 -20 % Growth Inhibition % Growth Inhibition 125 250 500 125 250 500 15.6 31.6 62.5 15.6 31.6 62.5 -40 1000 2000 -40 1000 2000 -60 -60 Concentration (nM) Concentration (nM)

D10 cmyc-10 cmyc-50 GFP-10 GFP-50 D10 cmyc-10 cmyc-50 GFP-10 GFP-50

2-ASA-9 2-ASA-14 120 120 100 100 80 80 60 60 40 40 20 20 0 0 -20 -20 % Growth Inhibition % Growth Inhibition 125 250 500 125 250 500 15.6 31.6 62.5 15.6 31.6 62.5 -40 1000 2000 -40 1000 2000 -60 -60 Concentration (nM) Concentration (nM)

D10 cmyc-10 cmyc-50 GFP-10 GFP-=50 D10 cmyc-10 cmyc-50 GFP-10 GFP-50

P. falciparum IC50 (nM) [SD] Compound D10 cmyc-10 cmyc-50 GFP-10 GFP50

QNE 65 [42] 57 [32] 64 [21] 67 [23] 65 [21]

SAHA 19 [5] 17 [6] 14 [3] 16 [3] 14 [4]

2-ASA-9 26 [2] 27 [3] 23 [6] 20 [5] 18 [4]

2-ASA-14 16 [1] 14 [7] 15 [3] 12 [4] 11 [3]

Figure 6.10 Growth inhibition activities of D10 PfHDAC1 transgenic versus D10 wildtype parasites. Growth inhibition was determined using an isotopic micro titre test as previous described (Section 2.17) for the antimalarial quinine (QNE), SAHA, 2-ASA-9, and 2-ASA-14. D10 wildtype (D10), D10 transfected with the pHH1-PFI1260c-cmyc plasmid maintained on 10nM and 50nM WR99210 (cmyc-10 and cmyc-50, respectively), and D10 transfected with the pHH1-PFI1260c-GFP plasmid maintained on 10nM and 50nM WR99210 (GFP-10 and GFP-50, respectively) are shown. Mean % growth inhibition are shown for each compound carried out on five separate occasions. Error bars indicate the standard deviation of the five experiments. Mean IC50 values [SD)] are summarised in the Table. 110

Chapter 6

6.3.2.6 Comparative deacetylase activities of D10 wildtype versus PfHDAC1-cmyc

parasites

Total deacetylase activity of nuclear and cytosolic extracts from PfHDAC1-cmyc mutant and

D10 wildtype parasites was examined to determine whether overexpression of PfHDAC1 resulted in an increase in enzyme activity in mutant parasites. Western blots analysis shows that

PfHDAC1 protein is present more abundantly in nuclear extracts compared to cytosolic extracts in both D10 and transgenic parasite lines (Figure 6.11A). This correlated well with the relative deacetylase activities observed in cytosolic extracts and nuclear extracts (Figure 6.11B). A comparison of total deacetylase from cytosolic and nuclear extracts found that there was no significant difference in the total deacetylase activities between D10 and D10 PfHDAC1 mutant parasites (P > 0.1; Figure 6.11B). Sensitivity of nuclear extracts to the HDAC inhibitor TSA (at

1µM) was similar for both D10 and mutant parasites (75% and 72% deacetylase inhibition for

D10 and mutant parasites, respectively; Figure 6.11C).

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A D10 cmyc B 12 3 4 12 3 4 kDa Cytosolic (cytosolic) 9000 55- anti-PfHDAC1 6000 43-

AFU 3000

0 55- (nuclear) 1234 anti-PfHDAC1 Extracts 43-

Nuclear (nuclear) 9000 34- anti-GAPDH 6000

C AFU 3000 0 9000 1234 Extracts

6000 Average DeacetylaseActivity 9000 AFU

3000 6000

AFU 3000

0 cytosolic nuclea r 0 D10 D10 + TSA cmyc cmyc + TSA Extracts

Figure 6.11 Comparative deacetylase activity in D10 wildtype versus D10 PfHDAC1-cmyc overexpression parasites. (A) Western blot analysis of cytosolic and nuclear protein extracts probed with anti-PfHDAC1 anti-sera, and anti-GAPDH anti-sera (a kind gift from Dr Daubenberger, Swiss Tropical Institute) as protein loading control. (B) Deacetylase activity expressed as arbitrary fluorescence units (AFU) of ~1 × 108 parasites equivalents were used per reaction. D10 is shown in black bars and PfHDAC1-cmyc (grown in the presence of 50nM WR99210) is shown in white bars. In each case, 1 – 4 represents independent extracts from each parasite line. Average deacetylase activities of the cytosolic and nuclear extracts plus standard deviation (SD) are shown. (C) Deacetylase activity of D10 and cmyc nuclear extracts (black bars) or treated with 1µM TSA (white bars) of ~1 × 108 parasites equivalent were used per reaction. Mean deacetylase activities (arbitrary fluorescent units; AFU) of four independent extracts (SD) are shown.

6.4 Discussion

In metazoan organisms HDAC1 and HDAC2 are homologues of yeast Rpd3 and are essential in embryonic development (reviewed in Brunmeir et al., 2009). As discussed in Chapter 5.3.1, all Plasmodium parasites species contain one copy of a gene whose open reading frame is homologous to the yeast Rpd3 – termed PfHDAC1 in P. falcipaurm. To investigate whether

PfHDAC1 is essential to parasite development, a single homologous crossover recombination strategy was employed to attempt genetically disrupt Pfhdac1. Although parasite populations were able to be positively selected on WR99210, PCR analysis of transfected parasite lines 112

Chapter 6 indicated that integration of the disruption plasmid into the Pfhdac1 locus did not take place

(Figure 6.2) suggesting this gene is essential in P. falciparum. Failure to disrupt Pfhdac1 was also recently communicated to us by another group (Personal communication, Alan Cowman,

WEHI). However, to confirm that pfhdac1 is essential to the parasite, various inducible knockdown strategies might also be used in future studies. Recently, the growth of in vitro cultured P. falciparum parasites was shown to be reduced by 50% following 48 h incubation with a double-stranded RNA molecule directed against the pfhdac1 gene (Sriwilaijaroen et al.,

2009). However, the mechanism of silencing by this double-stranded RNA approach is not clear. A study by Baum et al, (2009) demonstrated that the P. falciparum genome does not appear to possess the conventional Dicer and Argonaute machineries necessary for RNA mediated interference. A possible explanation for the apparent double-stranded RNA knockdown of Pfhdac1 is that it is mediated by an antisense phenomenon (Militello et al.,

2008). A study by Teusher et al, (2007) has demonstrated cellular damages to in vitro cultured

P. falciparum infected erythrocytes transfected with an antisense plasmid targeting the P. falciparum M18 aspartyl aminopeptidase. Another approach that may be useful for analysing essential genes in this parasite is the use of the T. gondii tetracycline repressor-based inducible knockdown system (Meissner et al., 2001). This system has been shown to regulate inducible expression of the GFP reporter in P. falciparum parasites (Meissner et al., 2005). While inducible knockdowns have potential for studying functionally essential genes in P. falciparum, this approach has not yet found broad applicability in malaria parasite research. An alternative approach which also has potential in the study of essential genes in P. falciparum parasite is the

FKBP destabilization domain which can modulate expression of P. falciparum proteins

(Armstrong & Goldberg, 2007). This approach has been used to regulate the amount of a reporter protein (yellow fluorescent protein) produced in P. falciparum infected erythrocytes

(Armstrong & Goldberg, 2007). This approach has also been shown to be effective in reversing phenotypic properties (swollen food vacuole) in a falcipain-2 knockout mutant (Armstrong &

Goldberg, 2007). However, one of the limitations of this system that needs to be considered is that leaky expression can occur whereby low level of degradation can produce a lethal outcome

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Chapter 6 for certain genes. Like the tetracycline inducible knockdown system, this FKBP destabilization inducible knockdown approach is not yet routinely used in malaria parasite research.

A second transgenic approach used in this study was to overexpress of P. falciparum HDAC1 to determine if this resulted in a change in sensitivity to HDAC inhibitors. This approach has been recently applied in the study of two P. falciparum aminopeptides to study their potential new antimalarial drug targets (Gardiner et al., 2006, Teuscher et al., 2007). Northern blot and

Western blot analysis showed that parasites transfected with plasmid containing the Pfhdac1 gene both transcribed and translated the fusion proteins in intra-erythrocytic stage parasites

(Figure 6.4 and Figure 6.5). However, IFA failed to detect PfHDAC1-cmyc and PfHDAC1-GFP protein in transgenic mutants. One possible explanation for this observation is that these tagged proteins are not being transported correctly (GFP live cytosol, Figure 6.7) and as a result are soluble and are being lost during fixation. Using alternative fixing methods such as paraformaldehyde may help overcome this problem and demonstrate that the tagged proteins are there but not in their correct location. The intra-erythrocytic growth of D10 PfHDAC1 transgenic parasites appeared to similar to D10 wildtype parasites (Figure 6.8 and Figure 6.9) indicating that overexpression of PfHDAC1 did not alter growth dynamics in intra-erythrocytic stage parasites.

Overexpression mutants were examined for any change in drug sensitivity to HDAC inhibitors.

There was no difference in drug sensitivity profile to the HDAC inhibitors tested in transgenic compared to D10 wildtype parasites (Figure 6.10). This was also the case when parasites were grown at higher WR99210 concentration (50nM; 10× the concentration used in positive selection of parasites) to increase episomal expression of PfHDAC1 (Figure 6.10). This may be due to the down regulation of endogenous PfHDAC1 in the mutant parasites as observed by

Western blot in D10 PfHDAC1-cmyc mutants (Figure 6.11A) or incorrect localization as discussed above. As would be expected, there was no differences in the total deacetylase activity of both the cytosolic and nuclear extracts combined in D10 wildtype and mutant

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Chapter 6 parasites. The deacetylase activity of these extracts was confirmed using the HDAC inhibitor

TSA (Figure 6.11C).

The apparent down-regulation of PfHDAC1 in the overexpressing mutants is interesting and suggests that the expression of this enzyme is tightly regulated. These data also indicate that while perhaps not correctly localised that the transgenic protein is functional. A role in gene regulation, as anticipated for HDAC enzymes, is likely to require tight regulation and as a result their down regulation in order to compensate for the transgenic PfHDAC1 is not surprising. To our knowledge, this appears to be the first report of this tight regulation of gene expression in P. falciparum. While, in T. gondii (an apicomplexan parasite related to P. falciparum) parasites stable expression the class I homologue TgHDAC3 as a HA-FLAG fusion has been reported

(Saksouk et al., 2005). The relative enzyme activity produced by the TgHDAC3-HA-FLAG transgenic parasites as compared to wildtype parasites was however not examined. In mice, studies have shown overexpression of HDAC3 (a class I homologue) in cardiac myocytes results in a 4 – 8 fold higher protein levels. Moreover, it appears that the enzyme activity of transgenic cells is only 2 fold greater than in wildtype cells (Trivedi et al., 2008). In addition, overexpression of HDAC3 did not change expression of the other class I homologues (HDAC1 and HDAC2) in these transgenic cells. The modest increase in enzyme activity in these transgenic cells underscores the complex regulation of HDAC enzymes and mirrors well with the down-regulation of these enzymes in P. falciparum parasites as observed in this study.

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Chapter 7

General discussion and future directions

In this study, a panel of fourteen non-peptidic hydroxamate-based HDAC inhibitors derived from L-CYS (six compounds) and 2-ASA (eight compounds) were shown to have potent antimalarial activity against P. falciparum in vitro (IC50 13 – 339nM). One 2-ASA compound

(2-ASA-9) demonstrated in vivo antimalarial potential in mouse malaria model. However, treatment with this compound (10mg/kg, twice daily for three days) did not result in a cure in mice which may be due to the relatively short half life of this class of compound as demonstrated by the rapid clearance of another representative compound from the 2-ASA series

(2-ASA-14, T½ < 2h) following a single oral dose of 5mg/kg. In vitro metabolic stability data suggest hydrolysis of the hydroxamic acid zinc binding group is partly responsible the reduced in vivo antimalarial activity of this class of compound. Thus, in future studies, one possible focus will be the replacement of the hydroxamic acid with a more stable zinc binding group.

However, a potential challenge posed in finding suitable zinc binding groups is the relative narrow active site in PfHDAC enzymes as predicted in the PfHDAC1 homology model. There is also a very limited choice of zinc binding group available with demonstrated metabolic stability. The recent availability of functional recombinant PfHDAC1 (Patel et al., 2009) does however represent an important tool for rational design of HDAC inhibitors. The rPfHDAC1 will be a useful tool to rapidly screen new HDAC inhibitors with improved potency and selectivity for PfHDAC enzymes. HDAC inhibitors that are more parasite-selective are highly desirable as these HDAC inhibitors are more likely to selectively destroy malaria parasites with significantly reduced toxicity to the host.

Just how effective HDAC inhibitors might be in the field to treat malaria is still unknown.

However, it has been proposed that these compounds might provide an alternative or adjunct to artemisinin for the treatment of uncomplicated malaria even if current artemisinin combination therapies (ACTs) succeed (Dow et al., 2008). There is currently very limited combination data 116

Chapter 7 on the interactions of HDAC inhibitors and antimalarial drugs. In this study, in vitro isobologram studies demonstrated in vitro antagonistic interactions between the HDAC inhibitor 2-ASA-9 and the antimalarial drugs chloroquine or artemisinin. In contrast, a more promising interaction between quinine and the HDAC inhibitors SAHA or 2-ASA-9 was observed and this warrants further investigation. However, in vitro data must also be treated with caution. In study by Dow et al (2008) the in vivo antimalarial efficacy of chloroquine and the HDAC inhibitor WR301801 was assessed in combination in a mouse malaria model and provided cures where WR301801 monotherapy did not. Taken together, these findings indicate

HDAC inhibitors may serve as a potential partner drug in new combination therapy. Therefore, future combination studies with HDAC inhibitors should include other antimalarial agents and new generation compounds with better parasite specificity profiles. These future combination studies would be of particular interest, as there is recent evidence of the declining efficacy of the

ACTs (Dondorp et al., 2009, Noedl et al., 2008, Wongsrichanalai & Meshnick, 2008).

Another important aspect to improve the antimalarial potential of HDAC inhibitors is to better understand the mode of action of these compounds on parasites. In this study, different hydroxamate-based HDAC inhibitors were shown to cause histone hyperacetylation in P. falciparum infected erythrocytes and inhibit deacetylase activity in parasite nuclear extracts.

While these bioassays remain useful for screening HDAC inhibitors, however, they provide limited information about the mode of action of HDAC inhibitors and only useful as an indirect marker of HDAC inhibition. In addition to HDAC inhibition, the effect of HDAC inhibitors on parasite transcription was confirmed by Northern blot and microarray studies carried out in parallel with this study. Transcription data will be useful to study parasite-specific changes and to identify genes that could be use as biomarkers to screen for new parasite-selective compounds.

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Supplementary Figures

PfHDAC1 ------PvHDAC1 ------PkHDAC1 ------PbHDAC1 ------PyHDAC1 ------PcHDAC1 ------TgHDAC3 ------ChRpd3A ------CpRpd3A ------ChRpd3B ------CpRpd3B ------HsHDAC1 ------HsHDAC2 MRSPPCGLLRWFGGPLLASWCRCHLRFRAFGTSAGWYRAFPAPPPLLPPACPSPRDYRPH 60 HsHDAC3 ------HsHDAC8 ------MmHdac1 ------DrHdac1 ------DmRpd3 ------ScRpd3 ------AaHDLP ------

PfHDAC1 ------MSNRKKVAYFHDPDIGSYYYG 21 PvHDAC1 ------MSNRKKVAYFHDPDIGSYYYG 21 PkHDAC1 ------MSNRKKVAYFHDPDVGSYYYG 21 PbHDAC1 ------MSNRKKVAYFHDPDIGSYYYG 21 PyHDAC1 ------MSNRKKVAYFHDPDIGSYYYG 21 PcHDAC1 ------MSNRKKVAYFHDPDIGSYYYG 21 TgHDAC3 ------MALSALRKRVAYFYDPDIGSYYYG 24 ChRpd3A ------MGAKKKIAYFYDEEVGNFHYG 21 CpRpd3A ------RYLLYKLSMGAKKKIAYFYDEEVGNFHYG 29 ChRpd3B ------MAKRISYFYDGDIGSYYYG 19 CpRpd3B ------ILLVSHLNNIVVISLKMAKRVSYFYDGDIGSYYYG 35 HsHDAC1 ------MAQT-QGTRRKVCYYYDGDVGNYYYG 25 HsHDAC2 VSLSPFLSRPSRGGSSSSSSSRRRSPVAAVAGEPMAYSQGGGKKKVCYYYDGDIGNYYYG 120 HsHDAC3 ------MAKTVAYFYDPDVGNFHYG 19 HsHDAC8 ------MEEPEEPADSGQSLVPVYIYSPEYVSMCDS 30 MmHdac1 ------MAQT-QGTKRKVCYYYDGDVGNYYYG 25 DrHdac1 ------MALSSQGTKKKVCYYYDGDVGNYYYG 26 DmRpd3 ------MQSHSKKRVCYYYDSDIGNYYYG 23 ScRpd3 ------MVYEATPFDPITVKPSDKRRVAYFYDADVGNYAYG 35 AaHDLP ------MKKVKLIGTLDYGKYRYP 18

PfHDAC1 AGHPMKPQRIRMTHSLIVSYNLYKYMEVYRPHKSDVNELTLFHDYEYIDFLSSISLENYR 81 PvHDAC1 AGHPMKPQRIRMTHSLIVSYNLYKYMEVYRPHKSDVNELTLFHDYEYVDFLSSISMENYR 81 PkHDAC1 AGHPMKPQRIRMTHSLIVSYNLYKYMEVYRPHKSDVNELTLFHDYEYVDFLSSISMENYR 81 PbHDAC1 AGHPMKPQRIRMTHSLIVSYNLYKYMEVYRPHKSDVNELTLFHDYEYVDFLSSISMENYR 81 PyHDAC1 AGHPMKPQRIRMTHSLIVSYNLYKYMEVYRPHKSDVNELTLFHDYEYVDFLSSISMENYR 81 PcHDAC1 AGHPMKPQRIRMTHSLIVSYNLYKYMEVYRPHKSDVNELTLFHDYEYIDFLSSISMENYR 81 TgHDAC3 PGHPMKPQRIRMAHALVLSYDLYKHMEVYRPHKSIEPELCLFHSSDYISFLSSVSPENYK 84 ChRpd3A LGHPMKPHRVRMTHDLVSQYGLLEKVDVMVPTPGTVESLTRFHSNDYVDFLRSVNTDNMH 81 CpRpd3A LGHPMKPHRVRMTHDLVSQYGLLEKVDVMVPTPGTVESLTRFHSNDYVDFLRSVNTDNMH 89 ChRpd3B PGHPMKPQRIRMAHNLILSYDLYKHMEIYKPHKSPQSELVYFHEEDYINFLSSINPDNSK 79 CpRpd3B PGHPMKPQRIRMAHNLILSYDLYKHMEIYKPHKSPQSELVYFHEEDYINFLSSINPDNSK 95 HsHDAC1 QGHPMKPHRIRMTHNLLLNYGLYRKMEIYRPHKANAEEMTKYHSDDYIKFLRSIRPDNMS 85 HsHDAC2 QGHPMKPHRIRMTHNLLLNYGLYRKMEIYRPHKATAEEMTKYHSDEYIKFLRSIRPDNMS 180 HsHDAC3 AGHPMKPHRLALTHSLVLHYGLYKKMIVFKPYQASQHDMCRFHSEDYIDFLQRVSPTNMQ 79 HsHDAC8 LAK--IPKRASMVHSLIEAYALHKQMRIVKPKVASMEEMATFHTDAYLQHLQKVSQEGDD 88 MmHdac1 QGHPMKPHRIRMTHNLLLNYGLYRKMEIYRPHKANAEEMTKYHSDDYIKFLRSIRPDNMS 85 DrHdac1 QGHPMKPHRIRMTHNLLLNYGLYRKMEIYRPHKANAEEMTKYHSDDYIKFLRSIRPDNMS 86 DmRpd3 QGHPMKPHRIRMTHNLLLNYGLYRKMEIYRPHKATADEMTKFHSDEYVRFLRSIRPDNMS 83 ScRpd3 AGHPMKPHRIRMAHSLIMNYGLYKKMEIYRAKPATKQEMCQFHTDEYIDFLSRVTPDNLE 95 AaHDLP KNHPLKIPRVSLLLRFLDAMNLIDEKELIKSRPATKEELLLFHTEDYINTLMEAER-CQC 77

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PfHDAC1 EFTYQLKRFNVGEATDCPVFDGLFQFQQSCAGASIDGASKLNHHCADICVNWSGGLHHAK 141 PvHDAC1 EFTYQLKRFNVGEATDCPVFDGLFQFQQSCAGASIDGAAKLNHHCADICVNWSGGLHHAK 141 PkHDAC1 DFTCQLKRFNVGEATDCPVFDGLFQFQQSCAGASIDGASKLNHHCADICVNWSGGLHHAK 141 PbHDAC1 DFTYQLKRFNVGEATDCPVFDGLFQFQQSCAGASIDGAAKLNHHCADICVNWSGGLHHAK 141 PyHDAC1 DFTYQLKRFNVGEATDCPVFDGLFQFQQSCAGASIDGAAKLNHHCADICVNWSGGLHHAK 141 PcHDAC1 DFTYQLKRFNVGEATDCPVFDGLFQFQQSCAGASIDGAAKLNHHCADICVNWSGGLHHAK 141 TgHDAC3 EFSLQLKNFNVGEATDCPVFDGLFTFQQACAGASIDAAKKLNHHQADICVNWSGGLHHAK 144 ChRpd3A DYSDHLARFNVGE--DCPVFDGLWEFCQLSAGGSLGGAQSVNELGYQYAINWAGGLHHGK 139 CpRpd3A DYSDHLARFNVGE--DCPVFDGLWEFCQLSAGGSLGGAQSVNELGYQYAINWAGGLHHGK 147 ChRpd3B DFGLQLKRFNLGETTDCPVFDGLFEFQQICAGGSIDGAYKLNNEQSDICINWSGGLHHAK 139 CpRpd3B DFGLQLKRFNLGETTDCPVFDGLFEFQQICAGGSIDGAYKLNNEQSDICINWSGGLHHAK 155 HsHDAC1 EYSKQMQRFNVGE--DCPVFDGLFEFCQLSTGGSVASAVKLNKQQTDIAVNWAGGLHHAK 143 HsHDAC2 EYSKQMQRFNVGE--DCPVFDGLFEFCQLSTGGSVAGAVKLNRQQTDMAVNWAGGLHHAK 238 HsHDAC3 GFTKSLNAFNVGD--DCPVFPGLFEFCSRYTGASLQGATQLNNKICDIAINWAGGLHHAK 137 HsHDAC8 DHPDS-IEYGLGY--DCPATEGIFDYAAAIGGATITAAQCLIDGMCKVAINWSGGWHHAK 145 MmHdac1 EYSKQMQRFNVGE--DCPVFDGLFEFCQLSTGGSVASAVKLNKQQTDIAVNWAGGLHHAK 143 DrHdac1 EYSKQMQRFNVGE--DCPVFDGLFEFCQLSAGGSVAGAVKLNKQQTDIAINWAGGLHHAK 144 DmRpd3 EYNKQMQRFNVGE--DCPVFDGLYEFCQLSAGGSVAAAVKLNKQASEICINWGGGLHHAK 141 ScRpd3 MFKRESVKFNVGD--DCPVFDGLYEYCSISGGGSMEGAARLNRGKCDVAVNYAGGLHHAK 153 AaHDLP VPKGAREKYNIGG-YENPVSYAMFTGSSLATGSTVQAIEEFLKG--NVAFNPAGGMHHAF 134

PfHDAC1 MSEASGFCYINDIVLGILELLKY-HARVMYIDIDVHHGDGVEEAFYVTHRVMTVSFHKFG 200 PvHDAC1 MSEASGFCYINDIVLGILELLKY-HARVMYIDIDVHHGDGVEEAFYVTHRVMTVSFHKFG 200 PkHDAC1 MSEASGFCYINDIVLGILELLKY-HARVMYIDIDVHHGDGVEEAFYVTHRVMTVSFHKFG 200 PbHDAC1 MSEASGFCYINDIVLGILELLKY-HARVMYIDIDVHHGDGVEEAFYVTHRVMTVSFHKFG 200 PyHDAC1 MSEASGFCYINDIVLGILELLKY-HARVMYIDIDVHHGDGVEEAFYVTHRVMTVSFHKFG 200 PcHDAC1 MSEASGFCYINDIVLGILELLKY-HARVMYIDIDVHHGDGVEEAFYVTHRVMTVSFHKFG 200 TgHDAC3 RSEASGFCYINDIVLGILELLKY-HARVMYIDIDIHHGDGVEEAFYVSHRVMTVSFHKFG 203 ChRpd3A KHEASGFCYVNDCVLGALEFLKY-QHRVCYVDIDIHHGDGVEEAFYTSPRCMCVSFHKYG 198 CpRpd3A KHEASGFCYVNDCVLGALEFLKY-QHRVCYVDIDIHHGDGVEEAFYTSPRCMCVSFHKYG 206 ChRpd3B RSEASGFCYINDIVLGILELLKY-HARVMYIDIDVHHGDGVEEAFYLSHRVLTVSFHKFG 198 CpRpd3B RSEASGFCYINDIVLGILELLKY-HARVMYIDIDVHHGDGVEEAFYLSHRVLTVSFHKFG 214 HsHDAC1 KSEASGFCYVNDIVLAILELLKY-HQRVLYIDIDIHHGDGVEEAFYTTDRVMTVSFHKYG 202 HsHDAC2 KSEASGFCYVNDIVLAILELLKY-HQRVLYIDIDIHHGDGVEEAFYTTDRVMTVSFHKYG 297 HsHDAC3 KFEASGFCYVNDIVIGILELLKY-HPRVLYIDIDIHHGDGVQEAFYLTDRVMTVSFHKYG 196 HsHDAC8 KDEASGFCYLNDAVLGILRLRRK-FERILYVDLDLHHGDGVEDAFSFTSKVMTVSLHKFS 204 MmHdac1 KSEASGFCYVNDIVLAILELLKY-HQRVLYIDIDIHHGDGVEEAFYTTDRVMTVSFHKYG 202 DrHdac1 KSEASGFCYVNDIVLAILELLKY-HQRVLYIDIDIHHGDGVEEAFYTTDRVMTVSFHKYG 203 DmRpd3 KSEASGFCYVNDIVLGILELLKY-HQRVLYIDIDVHHGDGVEEAFYTTDRVMTVSFHKYG 200 ScRpd3 KSEASGFCYLNDIVLGIIELLRY-HPRVLYIDIDVHHGDGVEEAFYTTDRVMTCSFHKYG 212 AaHDLP KSRANGFCYINDPAVGIEYLRKKGFKRILYIDLDAHHCDGVQEAFYDTDQVFVLSLHQSP 194

PfHDAC1 DY-FPGTG-DITDVGVNHGKYYSVNVPLNDGMTDDAFVDLFKVVIDKCVQTYRPGAIIIQ 258 PvHDAC1 DY-FPGTG-DITDIGVHHGKYYSVNVPLNDGITDDAFVDLFKVVIDKCVQTYKPGAIILQ 258 PkHDAC1 DY-FPGTG-DITDIGVHHGKYYSVNVPLNDGITDDAFVDLFKAVIDKCVQTYRPGAIILQ 258 PbHDAC1 DY-FPGTG-DITDVGVNHGKYYSVNVPLNDGITDEAFVDLFKVVIDKCVQSYKPGAIILQ 258 PyHDAC1 DY-FPGTG-DITDVGVNHGKYYSVNVPLNDGITDEAFVDLFKVVIDKCVQSYKPGAIILQ 258 PcHDAC1 DY-FPGTG-DITDVGVNHGKYYSVNVPLNDGITDEAFVDLFKVVIDKCVQSYKPGAIILQ 258 TgHDAC3 DF-FPGTG-DVTDVGASQGKYYAVNVPLNDGMDDDSFVALFKPVITKCVDVYRPGAIVLQ 261 ChRpd3A DY-FPGTG-ALNDVGVEEGLGYSVNVPLKDGVDDATFIDLFTKVMTLVMENYRPGAIVLQ 256 CpRpd3A DY-FPGTG-ALNDVGVEEGLGYSVNVPLKDGVDDATFIDLFTKVMTLVMENYRPGAIVLQ 264 ChRpd3B EF-FPGTG-DITDIGVAQGKYYSVNVPLNDGIDDDSFLSLFKPIISKCIEVYRPGAIVLQ 256 CpRpd3B EF-FPGTG-DITDIGVAQGKYYSVNVPLNDGIDDDSFLSLFKPIISKCIEVYRPGAIVLQ 272 HsHDAC1 EY-FPGTG-DLRDIGAGKGKYYAVNYPLRDGIDDESYEAIFKPVMSKVMEMFQPSAVVLQ 260 HsHDAC2 EY-FPGTG-DLRDIGAGKGKYYAVNFPMRDGIDDESYGQIFKPIISKVMEMYQPSAVVLQ 355 HsHDAC3 NYFFPGTG-DMYEVGAESGRYYCLNVPLRDGIDDQSYKHLFQPVINQVVDFYQPTCIVLQ 255 HsHDAC8 PGFFPGTG-DVSDVGLGKGWYYSVNVPIQDGIQDEKYYQICESVLKEVYQAFNPKAVVLQ 263 MmHdac1 EY-FPGTG-DLRDIGAGKGKYYAVNYPLRDGIDDESYEAIFKPVMSKVMEMFQPSAVVLQ 260 DrHdac1 EY-FPGTG-DLRDIGAGKGKYYAVNYPLRDGIDDESYEAIFKPIMSKVMEMYQPSAVVLQ 261 DmRpd3 EY-FPGTG-DLRDIGAGKGKYYAVNIPLRDGMDDDAYESIFVPIISKVMETFQPAAVVLQ 258 ScRpd3 EF-FPGTG-ELRDIGVGAGKNYAVNVPLRDGIDDATYRSVFEPVIKKIMEWYQPSAVVLQ 270 AaHDLP EYAFPFEKGFLEEIGEGKGKGYNLNIPLPKGLNDNEFLFALEKSLEIVKEVFEPEVYLLQ 254

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PfHDAC1 CGADSLTGDRLGRFNLTIKGHARCVEHVRSYNIPLLVLGGGGYTIRNVSRCWAYETGVVL 318 PvHDAC1 CGADSLTGDRLGRFNLTIKGHARCVEHVRSYNLPLLVLGGGGYTIRNVSRCWAYETGVVL 318 PkHDAC1 CGADSLTGDRLGRFNLTIKGHARCVEHVRSYNIPLLVLGGGGYTIRNVSRCWAYETGVVL 318 PbHDAC1 CGADSLTGDRLGRFNLTIKGHARCVEHVRSYNLPLLVLGGGGYTIRNVSRCWAYETGVVL 318 PyHDAC1 CGADSLTGDRLGRFNLTIKGHARCVEHVRSYNLPLLVLGGGGYTIRNVSRCWAYETGVVL 318 PcHDAC1 CGADSLTGDRLGRFNLTIKGHARCVEHVRSYNLPLLVLGGGGYTIRNVSRCWAYETGVVL 318 TgHDAC3 CGADSLTGDRLGKFNLTIKGHAACVAFVKSLDIPLLVLGGGGYTIRNVARCWAYETGVVL 321 ChRpd3A CGADSLSGDRLGCFNLSLKGHGHAVSFLKKFNVPLLILGGGGYTLRNVPKCWTYETSLIV 316 CpRpd3A CGADSLSGDRLGCFNLSLKGHGHAVSFLKKFNVPLLILGGGGYTLRNVPKCWTYETSLIV 324 ChRpd3B CGADSVRGDRLGRFNLSIKGHAECVEFCKKFNIPLLILGGGGYTIRNVARTWAYETATIL 316 CpRpd3B CGADSVRGDRLGRFNLSIKGHAECVEFCKKFNIPLLILGGGGYTIRNVARTWAYETATIL 332 HsHDAC1 CGSDSLSGDRLGCFNLTIKGHAKCVEFVKSFNLPMLMLGGGGYTIRNVARCWTYETAVAL 320 HsHDAC2 CGADSLSGDRLGCFNLTVKGHAKCVEVVKTFNLPLLMLGGGGYTIRNVARCWTYETAVAL 415 HsHDAC3 CGADSLGCDRLGCFNLSIRGHGECVEYVKSFNIPLLVLGGGGYTVRNVARCWTYETSLLV 315 HsHDAC8 LGADTIAGDPMCSFNMTPVGIGKCLKYILQWQLATLILGGGGYNLANTARCWTYLTGVIL 323 MmHdac1 CGSDSLSGDRLGCFNLTIKGHAKCVEFVKSFNLPMLMLGGGGYTIRNVARCWTYETAVAL 320 DrHdac1 CGADSLSGDRLGCFNLTIKGHAKCVEYMKSFNLPLLMLGGGGYTIRNVARCWTFETAVAL 321 DmRpd3 CGADSLTGDRLGCFNLTVKGHGKCVEFVKKYNLPFLMVGGGGYTIRNVSRCWTYETSVAL 318 ScRpd3 CGGDSLSGDRLGCFNLSMEGHANCVNYVKSFGIPMMVVGGGGYTMRNVARTWCFETGLLN 330 AaHDLP LGTDPLLEDYLSKFNLSNVAFLKAFNIVREVFGEGVYLGGGGYHPYALARAWTLIWCELS 314

PfHDAC1 NKHHEMPDQISLN-DYYDYYAPDFQLHLQPSN-IPNYNSPEHLSRIKMKIAENLR--HIE 374 PvHDAC1 NKHHEMPDQISLN-DYYDYYAPDFQLHLQPSS-IPNYNSPEHLSRIKMKITENLR--NIE 374 PkHDAC1 NKHHEMPDQISLN-DYYDYYAPDFQLHLQPSS-IPNYNSPEHLSRIKIKIAENLR--NIE 374 PbHDAC1 NKHHEMSDQISLN-DYYDYYAPDFQLHLQPSS-IPNYNSPEHLSKIKMKITENLR--NIE 374 PyHDAC1 NKHHEMSDQISLN-DYYDYYAPDFQLHLQPSS-IPNYNSPEHLNKIKMKITENLR--NIE 374 PcHDAC1 NKHHEMSDQISLN-DYYDYYAPDFQLHLQPSS-IPNYNSPEHLSKIKMKIAENLR--NIE 374 TgHDAC3 DRHREMSPHVPLN-DYYDYYAPDFQLHLTPSS-IPNSNSPEHLEKIKTRVLSNLS--YLE 377 ChRpd3A D--TYIDEQLPNSSNFYGYYGPDFSLAVRTSN-MENLNSRQDCEEIYRKISENFRDYVFP 373 CpRpd3A D--TYIDEQLPNSSNFYGYYGPDFSLAVRTSN-MENLNSRQDCEEIYRKISENFRDYVFP 381 ChRpd3B DRTDLISDNIPLN-DYYDYFAPDFKLHIPPLN-LPNMNSPEHLEKIKAKVIDNLR--YLE 372 CpRpd3B DRTDLISDNIPLN-DYYDYFAPDFKLHIPPLN-LPNMNSPEHLEKIKAKVIDNLR--YLE 388 HsHDAC1 D--TEIPNELPYN-DYFEYFGPDFKLHISPSN-MTNQNTNEYLEKIKQRLFENLR--MLP 374 HsHDAC2 D--CEIPNELPYN-DYFEYFGPDFKLHISPSN-MTNQNTPEYMEKIKQRLFENLR--MLP 469 HsHDAC3 E--EAISEELPYS-EYFEYFAPDFTLHPDVSTRIENQNSRQYLDQIRQTIFENLK--MLN 370 HsHDAC8 G--KTLSSEIPDH-EFFTAYGPDYVLEITPSC-RPDRNEPHRIQQILNYIKGNLK----- 374 MmHdac1 D--TEIPNELPYN-DYFEYFGPDFKLHISPSN-MTNQNTNEYLEKIKQRLFENLR--MLP 374 DrHdac1 D--STIPNELPYS-DYFEYFGPDFKLHISPSN-MTNQNTNDYLEKIKQRLFENLR--MLP 375 DmRpd3 A--VEIANELPYN-DYFEYFGPDFKLHISPSN-MTNQNTSEYLEKIKNRLFENLR--MLP 372 ScRpd3 N--VVLDKDLPYN-EYYEYYGPDYKLSVRPSN-MFNVNTPEYLDKVMTNIFANLE--NTK 384 AaHDLP GREVPEKLNNKAKELLKSIDFEEFDDEVDRSYMLETLKDPWRGGEVRKEVKDTLEK---A 371

PfHDAC1 HAPGVQFSYVPPDFFNSDID----DESDKNQYELKDDSGGGRAPGTRAKEHSTTHHLRRK 430 PvHDAC1 HAPGVQFAYVPPDFFDSEID----DECEKNQYELKDDGGGGRAAGTRAKDHATSHHLRRK 430 PkHDAC1 HAPGVQFAYVPPDFFDSEID----DECDKNQYELKDDSGGGRAPGTRSKEHSTTHHLRRK 430 PbHDAC1 HAPGVQFSYVPPDFFDSDID----DKSDKNQYELKDDSGGGRAAGTRGKEHSSTHHLRRK 430 PyHDAC1 HAPGVQFSYVPPDFFDSDID----DKSDKNQYELKDDSGGGRAAGTRGKEHSSTHHLRRK 430 PcHDAC1 HAPGVQFSYVPPDFFDSDID----DKSDKNQYELKDDSGGGRAAGTRGKEHSSSHHLRRK 430 TgHDAC3 HAPGVQFAYVPPDFFGEDND----DEDEFMQNQVDNEGGGRAAGATAHTAANAPYRIRRK 433 ChRpd3A IGSQISAYDIPEKLPLLYNPNKTPDDYKDG---NNIKHEQHQDFDDEMKEWPTVDYNNRA 430 CpRpd3A IGSQISAYDIPEKLPLLYNPNKTPDDYKDG---NNIKHEQHQDFDDEMKEWPTVDYNNRA 438 ChRpd3B HAPGVEFAYVPSDFFDREASNLQKQEDEEREEELSSWHGGGRAAGS--TESQGNHNEKPK 430 CpRpd3B HAPGVEFAYVPSDFFDREASNLQKQEDEEREEELSSWQGGGRAAGS--TESQGNHNEKPK 446 HsHDAC1 HAPGVQMQAIPEDAIPEESGDEDEDDPDKR--ISICSSDKRIACEEEFSDSEEEGEGGRK 432 HsHDAC2 HAPGVQMQAIPEDAVHEDSGDEDGEDPDKR--ISIRASDKRIACDEEFSDSEDEGEGGRR 527 HsHDAC3 HAPSVQIHDVPADL-LTYDRTDEADAEERG---PEENYSR-PEAPNEFYDGDHDNDKESD 425 HsHDAC8 HVV------377 MmHdac1 HAPGVQMQAIPEDAIPEESGDEDEEDPDKR--ISICSSDKRIACEEEFSDSDEEGEGGRK 432 DrHdac1 HAPGVQMQAIPEDAVQEDSGDE-EDDPDKR--ISIRAHDKRIACDEEFSDSEDEGQGGRR 432 DmRpd3 HAPGVQIQAIPEDAINDESDDEDKVDKDDR--LPQSDKDKRIVPENEYSDSEDEGEGGRR 430 ScRpd3 YAPSVQLNHTPRDAEDLGDVEEDSAEAKDTKGGSQYARDLHVEHDNEFY------433 AaHDLP KASS------375

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PfHDAC1 NYDDDFFDLSDRDQSIVPY------449 PvHDAC1 NYEDDFFDLSDRDQNVAL------448 PkHDAC1 NYEDDFFDLSDRDQNIVL------448 PbHDAC1 NYEDDFFDMSDRDQGII------447 PyHDAC1 NYEDDFFDMSDRDQGII------447 PcHDAC1 NYEDDFFDMSDRDQGII------447 TgHDAC3 DYANDFEDMADRDQKVPI------451 ChRpd3A IG------432 CpRpd3A IG------440 ChRpd3B SSKKLQKEHASEFY------444 CpRpd3B SSRKLQKEHASEFY------460 HsHDAC1 NSSNFKK-AKRVKTEDEKEK---DPEEKKEVTEE-EKTKE---EKPEAKGVKEEVKLA-- 482 HsHDAC2 NVADHKKGAKKARIEEDKKE---TEDKKTDVKEE-DKSKDNSGEKTDTKGTKSEQLSNP- 582 HsHDAC3 VEI------428 HsHDAC8 ------MmHdac1 NSSNFKK-AKRVKTEDEKEK---DPEEKKEVTEE-EKTKE---EKPEAKGVKEEVKLA-- 482 DrHdac1 NAANYKK-PKRVKTEEEK-----DGEEKKDVKEE-EKASE---EKMDTKGPKEELKTV-- 480 DmRpd3 DNRSYKGQRKRPRLDKDTNSNKASSETSSEIKDEKEKGDGADGEESTASNTNSNNNSNNK 490 ScRpd3 ------AaHDLP ------

PfHDAC1 ------PvHDAC1 ------PkHDAC1 ------PbHDAC1 ------PyHDAC1 ------PcHDAC1 ------TgHDAC3 ------ChRpd3A ------CpRpd3A ------ChRpd3B ------CpRpd3B ------HsHDAC1 ------HsHDAC2 ------HsHDAC3 ------HsHDAC8 ------MmHdac1 ------DrHdac1 ------DmRpd3 SDNDAGATANAGSGSGSGSGAGAKGAKENNI 521 ScRpd3 ------AaHDLP ------

Figure S1. Multiple sequence alignment of class I HDAC amino acid sequences. Alignment of class I HDAC homologues from P. falciparum (PfHDAC1; CAD51938), P. vivax (PvHDAC1; XP_001614790), P. knowlesi (PkHDAC1; XP_002258827), P. yoelii (PyHDAC1; EAA15658), P. berghei (PbHDAC1; CAA98271), P. chabaudi (PcHDAC1; CAH87890),. T. gondii (TgHDAC3; AYY53803), C. parvum (CpRpd3A; XP_627509 and CpRpd3B; XP_625348), C. hominis (ChRpd3A; XP_668169 and ChRpd3B; XP_667698), human (HsHDAC1; CAG46518, HDAC2; NP_001518, HDAC3; NP_003874, and HDAC8; AAF73428), mouse (MmHDAC1; AAI08372), D. rerio (DrHDAC1; AAI65208), D. melanogaster (DmRpd3; NP_047918), S. cerevisiae (ScRpd3; AAB_00328), and A. aeolicus (AaHDLP; NP_213698). Full length amino acid sequences were used in alignment.

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HsHDAC4 --MSSQSHPDGLSGRDQPVELLNPARVNHMPSTVDVATALPLQVAPSAVPMDLRLDHQ-- 56 HsHDAC5 --MNSPNESDGMSGREPSLEILPRTSLHSIPVTVEVKPVLPRAMPSSMGGGGGGSPSP-- 56 PfHDAC3 --MKKKITNHKKKNENAKLNIDHDNSSDDNDKTPQIDNIFKNLIVDSYKSINDINNYMRQ 58 PfHDAC2 MDIVKDERNIRYEYELDKIKKKKKDNLLIMNILYNKINYICYILKSNHRDILNDINFENR 60 : . . . :: : : : .

HsHDAC4 ----FSLP-----VAEPALREQQLQQELLALKQKQQ-IQRQILIAEFQRQHEQLSRQHEA 106 HsHDAC5 ----VELRGALVGSVDPTLREQQLQQELLALKQQQQ-LQKQLLFAEFQKQHDHLTRQHEV 111 PfHDAC3 SNLLYNEKKSKKNKTHPLDSKNTTDRKYNKYRRNEQ-NMNKVINYDDKKNYDDIENYDNI 117 PfHDAC2 TAIEICLYYCKSYILFLLLFYPYLHNYIRILKNNDLRLYKNFYKYILKRRNNLHILLLKY 120 .. :.:: .:. ::. : :

HsHDAC4 QLHEHIKQQQEMLAMKHQQELLEHQR--KLERHRQ-----EQELEKQHREQKLQQLK--- 156 HsHDAC5 QLQKHLKQQQEMLAAKQQQEMLAAKRQQELEQQRQREQQRQEELEKQRLEQQLLILR--- 168 PfHDAC3 ENYDNIENYDNVENYDYKLPYDEDIIYDEKQNNSDVVIIGYKNFKKGKKKISLDSFNSE- 176 PfHDAC2 RILECMKLKDKDVNDSNTISYIDVYSMNRYMNDEDDIDKYYYKPKFSYKNNNMNNLLLLN 180 . . :: :: . . .. : : : : .: : HsHDAC4 ------NKEKGKESAVASTEVKMKLQEFVLNKKKALAHRN------LNHCISS 197 HsHDAC5 ------NKEKSKESAIASTEVKLRLQEFLLSKSKEPTPGG------LNHSLPQ 209 PfHDAC3 ------LSPQSNTSPIYIKKKINEINYHFIQKKKKKKNQKNGELNKKYSKKLSINKKNKK 230 PfHDAC2 VYFKDIMSKNNFICILFSRIYIKKYHKKIFRILLLILFYFSLPTFPFSKKFFITYHNWRD 240 . :. :: .:. . : .

HsHDAC4 DPRYWYGKTQHSSLDQSSPPQSG---VSTSYNHPVLGMYDAKDDFPLRKTASEPNLKLRS 254 HsHDAC5 HPKCWG--AHHASLDQSSPPQSGPPGTPPSYKLPLPGPYDSRDDFPLRKTASEPNLKVRS 267 PfHDAC3 YEKHKKYEKHKKYEKHKKYEKNKKYEKNKKYEKNKKYEKNKKNEKNKKNEKNKKREKNKS 290 PfHDAC2 TEKDKIKEENEENHTNTKGINNSTYDNIYRSVLEDKVEGKLIEHNKNISKNNVGVQAVQE 300 : :. :.. :. . :. . . :.

HsHDAC4 RLKQKVAERRSSP------LLRRKDGPVVTALKKRPL 285 HsHDAC5 RLKQKVAERRSSP------LLRRKDGTVISTFKKRAV 298 PfHDAC3 GLINNVKKIKRNKKSLSEENLNDSLFSNDEQVNENKYILDKLLISLKNNSDANIHKKKNF 350 PfHDAC2 GVVYGTEEILKNENKGTGGMQGNNIKKKKDRGKKKDMDEDFSFYCKRKYFTTPFFEHINI 360 : . : . : :. :: .

HsHDAC4 DVT------DSACSSAPGSG------PSSPNNSSGSVSAENGIAPAVP-- 321 HsHDAC5 EITGAGPGASSVCNSAPGSG------PSSPN-SSHSTIAENGFTGSVP-- 339 PfHDAC3 YIEYEGNDDFTSFNSKENPNDYFLCMDNDEINNKLNKNKMYSSSYSTTKKKYIYKSHKGL 410 PfHDAC2 PTIHINDYFCSGNKIFEREEFKVINE------KKNKKKKDNTNKYESNYDSKYDNNN 411 : ...... :

HsHDAC4 ----SIPAETSLAHRLVAREGSAAPLPLYTS--PSLPNITLGLPATG------PSAGT 367 HsHDAC5 ----NIPTEMLPQHRALPLDSSPNQFSLYTS--PSLPNISLGLQATVTVTNSHLTASPKL 393 PfHDAC3 YDNDYMFNNNIFNNKIYNNEMYNNKFNIYNHNIPSNKNYKTNLKGRMSLQMSHDISQEAL 470 PfHDAC2 SRDHYNNFRTYEENNIYNSDSSSSVSSSLSDDILSDIEDENMLKELFKNENKIYSKEESK 471 . :. : . * : * .

HsHDAC4 AGQ------QDAERLTLPALQQR--LS--LFPGTHLTPYLSTSPLERDGG-AAHS 411 HsHDAC5 STQ------QEAERQALQSLRQGGTLTGKFMSTSSIPGCLLGVALEGDGSPHGHA 442 PfHDAC3 NTRNYFE-NNKNFEKERLKKKMRKIRELNLLNNNVEYDCIGFVCDEEYMCEKLHFDENHV 529 PfHDAC2 KNDNEKERNIKNLMKEKQNILNEKLKDIDTNYSKSTLNDMKNDFFQMNIKEYDWYDDNNI 531 :: . ::: * :

HsHDAC4 P------LLQHMVLLEQPPAQAPLVTGL-GALPLHAQS-LVGADRVSPSIH------454 HsHDAC5 S------LLQHVLLLEQARQQSTLIA-----VPLHGQSPLVTGERVATSMRTVG--- 485 PfHDAC3 E------SPDRIKCIIKALKEKKLIN---KMVQIKCREALYDEIRECHSSTHINNIF 577 PfHDAC2 IRRVYRKEDDIIDNFTLKKEPFVSELWINVINFNVEDDNKNTLIHKACLVGNLNIIYILL 591 :.. :. . : : . :. * .

HsHDAC4 ------KLRQHRPLGRTQSAPLPQNAQ----ALQHLVIQQQH--QQFLEK 492 HsHDAC5 ------KLPRHRPLSRTQSSPLPQSPQ----ALQQLVMQQQH--QQFLEK 523 PfHDAC3 YSLKKKLKHNNQSVIYPFDKHDTYYTSYTGTVSIRAIGGLLNLCDVILCDKK--DKFKYI 635 PfHDAC2 YLNVDLFIYN---YKSELPVHCTIYNCDKYIFLLLLHNTIECIFYVLLEEYKRWNKTHNM 648 : * . . . : ::: : : ::

HsHDAC4 HKQQFQQQQLQMNKIIPKPSEPARQPESHPEETEEELREHQ-ALLDEPYLDRLPGQKEAH 551 HsHDAC5 QKQ----QQLQLGKILTKTGELPRQPTTHPEETEEELTEQQEVLLGEGALTMPREGSTES 579 PfHDAC3 DFKKSLRYNYDFYKNVNTNSIKRNYESNNMYHKKFLRRSKSESSIYRKCTTYDTFNDFAL 695 PfHDAC2 LNTKTESEKMKIQNRKNKLKEKKKNKKKNKNKKNKNKKNKNKKKINKSRINYNNKKKNNK 708 : .: : . . .: ..: .:. : . .

HsHDAC4 AQAGVQVKQEPIESDEEEAE-----PPREVEPGQRQ-PSEQELLFRQQALLLEQQRIHQL 605 HsHDAC5 ESTQEDLEEEDEEDDGEEEEDCIQVKDEEGESGAEEGPDLEEPGAGYKKLFSDAQPLQPL 639 PfHDAC3 LNHNKNVYKMNTYHEQIEKKNNNNIINSNNNNNNNNNNNINCSVYNYKNEIDVQNDIHNI 755 PfHDAC2 KNNQNVQNVQNVQNVQNVQNVQNVQNAQNNIDSFNSSEKHDEDIEEVNEMYKKYIKDGKF 768 . : : . .. . : : :

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HsHDAC4 RNYQAS---MEAAGIPVSFGGHRPLSRAQSSPASATFPVSVQEPPTKPRFTTGLVYDTLM 662 HsHDAC5 QVYQAP---LSLATVP-----HQALGRTQSSPAAPGGMKSPPDQPVKHLFTTGVVYDTFM 691 PfHDAC3 NNINMNNNIISEENNLDLEAADYNFGNEKDEHTNESYNEENNINQKKDYISQSNSSHNSY 815 PfHDAC2 FYYKNINDKFLYNVIQLYLSVIVKIIELGNFEFLKILFNYNSSIFSYIFLNKNMFSFLCS 828 : : : . . :. .

HsHDAC4 LKHQCTCGSSSSHPEHAGRIQSIWSRLQETGLRGKCECIRGRKATLEELQTVHSEAHTLL 722 HsHDAC5 LKHQCMCGNTHVHPEHAGRIQSIWSRLQETGLLSKCERIRGRKATLDEIQTVHSEYHTLL 751 PfHDAC3 YNEIHQGDSIDYNNNNINNDSAISNKLSICEKLSMDNKLSSISNNSNHQDILPNCFKNDE 875 PfHDAC2 IACMYNCLNVFIFYCGNIIKWSVFETYVEEQKLNFLERTKKRKNVSTYGTNNNDENQYNF 888 . :: . . : . . :

HsHDAC4 YGTNPLNRQKLDSKKLLGSLAS-VFVRLPCGGVGVDS------DTIWNEVHSAGAARL 773 HsHDAC5 YGTSPLNRQKLDSKKLLGPISQKMYAVLPCGGIGVDS------DTVWNEMHSSSAVRM 803 PfHDAC3 ENIHKDKNRDEIINCNNTNVIETEYLNKICENDNYTNNYEENY-KNNSFLNSNNSDIFNL 934 PfHDAC2 YDMNNEKKRNEFQNNNLFQTGPCSSIKKNKTNLDTSKDTSCFINSYEKIEDVHQQDFFNI 948 . :.:. : . . . : :. . .: HsHDAC4 AVGCVVELVFKVATG------ELKNGFAVVRPPGHHAEESTPMGFCYFNSVAVAAKLLQ 826 HsHDAC5 AVGCLLELAFKVAAG------ELKNGFAIIRPPGHHAEESTAMGFCFFNSVAITAKLLQ 856 PfHDAC3 PYRSYSSTVYSMDDCNDVNTFTDINCGFAAIRPPGHHCSRSHPSGFCIFNNISVACKYIF 994 PfHDAC2 LKCVPYIINNGRMSGIGNNMDDNNISGENNMSRENNMSRENNMSRENNMSSENNMSSENN 1008 : * : .: . .. :.. ..

HsHDAC4 QRLSVSKILIVDWDVHHGNGTQQAFYSDPSVLYMSLHRYDDGN------869 HsHDAC5 QKLNVGKVLIVDWDIHHGNGTQQAFYNDPSVLYISLHRYDNGN------899 PfHDAC3 KKYGIRKVFIFDWDVHHDNGTQEIFYGDKDVLCFSIHRFDKKVEEKNKKKRHVNKKKKKK 1054 PfHDAC2 MSSENNMSSENNMSSENNMSSENNISGENNISGENNISGENNISG------1053 : . .:. .::: : .: .: . :.

HsHDAC4 ------HsHDAC5 ------PfHDAC3 KYDIKKDDDNKKDDDNKKDDDNKKDGDNKKDDDNKIYGDNKIYDDNKKDDDNKIYGDNKK 1114 PfHDAC2 ------

HsHDAC4 ------FFPGSGAP 877 HsHDAC5 ------FFPGSGAP 907 PfHDAC3 DDDNKIYGDNKIYGDNKISNLEKNLKYEKTSNNYKKYKRKNKKSRKRYEENLFYPRTGAK 1174 PfHDAC2 ------ENNISGE 1060 ..

HsHDAC4 DEVGTGPGVGFNVNMAFTGGLDPPMGDAEYLAAFR-TVVMPIASEFAPDVVLVSSGFDAV 936 HsHDAC5 EEVGGGPGVGYNVNVAWTGGVDPPIGDVEYLTAFR-TVVMPIAHEFSPDVVLVSAGFDAV 966 PfHDAC3 NELGEKEGYKFNINVPLEKGYN----NCDVYYVFK-YLLLPILEKFRPEFIFISCGFDAS 1229 PfHDAC2 NNISGENNMSRENNMSRENNICYPNNMYNQNNMYNQNNMYNQNNMYNQNNMYYPNNMYNQ 1120 :::. . : *:. . : :. : : : : . .:

HsHDAC4 EGHPTPLGGYNLSARCFGYLTKQLMGLA----GGRIVLALEGGHDLTAICDASEACVSAL 992 HsHDAC5 EGHLSPLGGYSVTARCFGHLTRQLMTLA----GGRVVLALEGGHDLTAICDASEACVSAL 1022 PfHDAC3 IN--DPLGKCNLTHNLYQWMTFQLKHFANIFCNGRIILVLEGGYNLNYLPKCALACIKAL 1287 PfHDAC2 NNMYNQNNMYNPNNMYYQNNMYNQNNMY----NQNNMYYPNNMCNPNYLYNDNNNNLCNN 1176 . . . . : : : . . : :. : . : . :

HsHDAC4 LGNELDPLPEKVLQQRPNANAVRSMEKVMEIHSKYWRCLQRTTSTAGRSLIEAQTCENE- 1051 HsHDAC5 LSVELQPLDEAVLQQKPNINAVATLEKVIEIQSKHWSCVQKFAAGLGRSLREAQAGETE- 1081 PfHDAC3 IKKNKSTTTDKEFIKKLYQNNVNNKETNILNNQKECISNQDSSKRFLDAHEEKKKKNDEK 1347 PfHDAC2 MLYHYFKINNMVNHRFLNISNISFYDPHVNAILTCRAKDPIGTFCEMLYKKDNKRLIKN- 1235 : . : : . : : : . : : : :

HsHDAC4 ------EAETVTAMASLSVGVKPAEK------RPDEEPMEEEP 1082 HsHDAC5 ------EAETVSAMALLSVGAEQAQAAAAREHSPRPAEEPMEQEP 1120 PfHDAC3 NVNRCDNMNDAQHIHGSNNINDAQHIHGNNSMNDAQHINDNNSINDAQHINGNNSMNDAQ 1407 PfHDAC2 ------DIVKKCINKNNSIKIFFSKECFKHIFVPEPCDHPYERNK 1274 : . . :.. :

HsHDAC4 PL------1084 HsHDAC5 AL------1122 PfHDAC3 HINGNNSMNDAQHINGNNSMNDAQLINGSNNTNDMYDKHYLNNHNLDNNIDEKLKSNKTY 1467 PfHDAC2 LKNNIPENSTRLDVLISNKHGILNINTFAKFKVKCVDRKATVNDILRVHDVSYLKFIINK 1334

HsHDAC4 ------HsHDAC5 ------PfHDAC3 ESLVSTNCVQKNEFLNYPKNNFINKKNMSDHANFYKLKYSSYDKYENKDINYYFRRLHTN 1527 PfHDAC2 IKNFKMSDDDLMYFDDFLTKAKKNFKNYLLTNNGNENTGNQNNPIQKNEILQTFYLINKK 1394

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HsHDAC4 ------HsHDAC5 ------PfHDAC3 NNIVSRTKKLITSGLLHYSTYKVIKYFLNVLKGEPYHLDIELPTYNEFIKKKKLEGREIN 1587 PfHDAC2 SELENIMLIRMNNKFMGNRDLYIQNYLSNIHTPMHANLKDKPLVSLELKKILSDSDSEDN 1454

HsHDAC4 ------HsHDAC5 ------PfHDAC3 PERKIKIKINRSSEFRKNDYCFNSYEIMNSYMQHKLNKLKHINNISNKNLQDMFIKQKNF 1647 PfHDAC2 NNNDIENYEPEKYVYMENG------IRMNILTDEGLTEAANVLYINNSILYNNNIFFNNN 1508

HsHDAC4 ------HsHDAC5 ------PfHDAC3 KYTDSSTTISNISQSDLYLSNDDINYSSSCSSTCNRRKVILLNKLKHKINRGLNNIYNTS 1707 PfHDAC2 LLYNNNILMSEKNTDYPFFHNEDIVGTNKSATNLLIQEKLSSYNINVREKKKKKNKWN-- 1566

HsHDAC4 ------HsHDAC5 ------PfHDAC3 PNNNNNNNNNNNNNNNNYNNYNNYNNYNNYNNNMVHTEKKTYKKKRKNQINDINLNAPVQ 1767 PfHDAC2 --KDKIEKLLLVDNDTFVNKYSFNCALSASGVVLKAVDYVHKQKKIVNCGYEKLKKCKKC 1624

HsHDAC4 ------HsHDAC5 ------PfHDAC3 MNRTQIDTNEENENLNSNKNKSNRTIKTNQNKLLSYNLSNLQNINNINNTHIIPYADLEH 1827 PfHDAC2 KYCKKCENCKKCKNSETCKNCKNCKKCEYCKKCKKYRIQNNSIIPNNRKKIFCVVRPPGH 1684

HsHDAC4 ------HsHDAC5 ------PfHDAC3 INDDNHLESHNTSNENYINNYYMNCDLTNLKYNENQVLNTFNIYTKIKKGFIFFYGSGHR 1887 PfHDAC2 HLGTFGAAQFNLTDEDVAAGSQGFCILNNVAVGLAYAKYTYKKFERIAIIDFDVHHGNGT 1744

HsHDAC4 ------HsHDAC5 ------PfHDAC3 NQWILPVHNKITKIIKLCSESEAFFYAWLYLSCLKKINISRTKNNYTSILDNNETIENVS 1947 PfHDAC2 EQIIRNLGLKKLTVNEYIDIYSWKGWKDNNDKKNIFFSSVHAYDGYFYPGTGYDTVELEP 1804

HsHDAC4 ------HsHDAC5 ------PfHDAC3 IQLPFEEREHILCKQFIKFTVPCYHIFLKRNQINSIRQKQSKQCKDDNNNNNHNITNNTF 2007 PfHDAC2 YIINVTLKKNMTSLEFLNIFHSKILIHLYYFKPNLLFLSAGFDGHQLDYVNNGFVKKNTS 1864

HsHDAC4 ------HsHDAC5 ------PfHDAC3 MDYMKKTYIEWSSSKIFKNNESIQYNDKINDGTNNMIYNSSSNEKKNNVLENDNSLYP-- 2065 PfHDAC2 TYFYLTKLVLSLQNKLNFPIISVLEGGYNTSKDMASVFSLSVLEHVLSFYYNDISFFRKK 1924

HsHDAC4 ------HsHDAC5 ------PfHDAC3 ------LHINQNIQEEAEKDVEVEEEHKTAICLSNVLSSMRHPCVMDIKMGIRLYG 2115 PfHDAC2 EIKLKDLKKNIERMERYKNDLKKYKNDLKKYKDDLKVFEDYLKENKNLFENYKNDLKKHR 1984

HsHDAC4 ------HsHDAC5 ------PfHDAC3 DDCNEESIQKKIEKAKNRSCLSHGFHLTSLIGWSKKKKEPFFISKEDAHSIKNDDNF--- 2172 PfHDAC2 NDFKKHKNDLKIFKDDFKKYKDDFKIYCDFNNYYDLKKSCNYFSIYYDDFNKYYDNLNVH 2044

HsHDAC4 ------HsHDAC5 ------PfHDAC3 ---VQAFISYFTACDNIQLSILLIKKILIILEEMKIFFIEQNYFAFYGTSLLFVFDSDPS 2229 PfHDAC2 EHTNNNQTTLQSNTDKNNTSIETCNTNIIDKMKNKIKKSEKENKKIKETPTLYYPFICIG 2104

HsHDAC4 ------HsHDAC5 ------PfHDAC3 KKENEDNYSSKDKIEKNELTKNISLSNENMISNDEHNNSFYYKDKKNKLCDQDKINFEEI 2289 PfHDAC2 KKKIINMFERYFSVFKEKTEETQNLNLFYKLVEYNNFLKIYDLRNIDMKNKMNKFMQTHE 2164

HsHDAC4 ------HsHDAC5 ------PfHDAC3 LNFKLNIEKVFYESLSNEERNIILQNKLNQKIIKSVHVYIIDFAHASLNKNQNDEGFLLG 2349 PfHDAC2 AVLKNILLECNYDYSKIDPIILPSNCYFYELLN---YLKIKGISMKPKTPTKLEKKFFSA 2221

124

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HsHDAC4 ------HsHDAC5 ------PfHDAC3 IISLHRIMDKTIQRIKELYLSNTDVTNDTV 2379 PfHDAC2 INNPYDFNKIDLENCKRHPKKNFEFINFWN 2251

Figure S2. Multiple sequence alignment of class II HDAC homologues. Alignment of class II HDAC homologues from P. falciparum (PfHDAC2; XP_001348864 and PfHDAC3; XP_001347363) and human (HsHDAC4; NP_006028 and, HsHDAC5; NP_005465). Full length amino acid sequences were used in alignment.

125

Appendix

Supplementary Tables

Table S1. List of P. falciparum strains. Name Origin Resistance status Reference

Dd2 Indochina CQ, QNE, SDX, PYR (Wellems et al., 1988)

3D7 West Africa Sensitive strain (Walliker et al., 1987)

PH1 Philippines CQ (Chen et al., 2005)

K1 Thailand CQ, PYR (Burkot et al., 1984)

D10 Papua New Guinea Sensitive strain (Walliker et al., 1987) aCQ: chloroquine, QNE: quinine, SDX: sulfadoxine, PYR, pyrimethamine

Table S2. List of antibodies. Name Species Peptide/antigen Source Anti-tetra-acetyl Rabbit Tetra-acetylated histone H4 Upstate, USA Histone H4 peptide Anti-di-acetyl Rabbit Di-acetylated histone H3 peptide Upstate, USA Histone H3 Anti-PfHDAC1 Mouse PfHDAC1 residues 425 - 438 QIMR peptide synthesis facility Anti-PfAMA-1 Mouse PfAMA-1 Dr. Alan Saul, QIMR Anti-PfHSP70 rabbit PfHSP70 Prof. Geoff McFadden, WEHI Anti-RGS-His mouse Hexa-histidines Qiagen, USA Anti-GFP mouse Green Fluorescent Protein (A. Roche, Germany Victoria) Anti-cmyc mouse cmyc peptide (human) Sigma, USA Anti-PfGAPDH rabbit PfGAPDH Dr. Daubenberger, Swiss Tropical Institute Anti-mouse-HRP Goat Mouse IgG (H + L) Zymed, USA Anti-rabbit-HRP Goat Rabbit IgG (H + L) Zymed, USA

126

Appendix

Table S3. List of primers. Name Sequence 5’ – 3’ Trancription studies (Chapter 4)

Alpha-II-tubulin F_PFD1050w_ExonIII (forward) GTACCACGTTGTGTGTTCG R_PFD1050w_ExonIII (reverse) TCATTCATATCCCTCATCTTCTCC

Histone H4 histone H4-F (forward) ATGTCAGGAAGAGGTAAGG histone H4-R (reverse) AACTCCAAAACCATATAAAGTTCTTCC

Stage Specific expression studies (Chapter 5) Primer A ATGTCTAATAGAAAAAAGGTTGC Primer B TTAATATGGTACAATAGATTGATCC Primer C. GGatcgatTTCTACACATCTGGCATGACC Primer D CGgaattcAAGTTGCCTATTTCC Primer E CCGctcgagATTTAATACAACCCCCGTTTCGTAAGCC Primer F AAACATCATGAAATGCCTG Msp-1 Msp-1N1 GCAGTATTGACAGGTTATGG Msp-1N2 GATTGAAAGGTATTTGAC

Recombinant PfHDAC1 studies (Chapter 5) h1BAMHI_f CGGgatccgATGTCTAATAGAAAAAAGGTTGC h1HINDIII_r CCCaagcttTTAATATGGTACAATAGATTGATCC

PfHDAC1 “knockout” studies (Chapter 6) HDAC1-F-ArvII TTcctaggATGTCTAATAGAAAAAAGGTGC HDAC1-R-ClaI GGatcgatTTCTACACATCTGGCATGACC

Integration PCR studies (Chapter 6) primer a TTcctaggATGTCTAATAGAAAAAAGGTGC primer b GGatcgatTTCTACACATCTGGCATGACC Primer c TTAATATGGTACAATAGATTGATCC Primer d CGgaattcAAGTTGCCTATTTCC Primer e CCGctcgagATTTAATACAACCCCCGTTTCGTAAGCC Primer f TGCCCTTCTCCTCCTGGAC Primer g ACATTTATGTGCACACATTCC Primer h AATGGTTTCTTAGACGTCAGGTGGC Primer i GTAAGGAGAAAATACCGCATC

PfHDAC1 “overexpression” studies (Chapter 6) HDAC1-F-BglII GGctgtctATGTCTAATAGAAAAAAGG HDAC1-R-PstI AActgcagATATGGTACAATAGATTGATCC

GFP GFP-F-BglII agatctATGAGTAAAGGAGAAGAACTTTTC GFP-R-NsiI atgcatGTCTGGATTATTTGTATAGTT 127

Appendix

Table S4. List of hits to C-terminus of PfHDAC1 (PFI1206c) % Gene length similarity/identity Gene IDa (nucleotide) to C-terminus of PfHDAC1 bPFI1260c 1350 93 PF14_0619 1131 57 MAL8P1.114 4404 59 PF14_0471 2148 59 PFE0980c 7467 58 PF14_0278 4323 59 PF13_0239 3735 58 PFD0585c 5787 59 PFB0460c 7722 58 PFF1380c 951 57 PFL1650w 6102 58 PF14_0428 3399 57 PF07_0107 2417 56 MAL8P1.34 3375 60 PF14_0058 4086 58 PF10_0363 2238 57 a Plasmodium Genome Resource (Aurrecoechea et al., 2009)

128

Appendix

Medias and Buffers

Luria Broth (LB)

10g Trytone 5g Yeast extract 10g NaCl 1L Deionised water

10× TAE

48.4g Tris-HCl 11.42mL Glacial Acetic acid 20mL 0.5M EDTA Add deionised water to 1L

4× SDS-PAGE Sample Buffer

4mL Glycerol 0.8g SDS 2.5mL 1M Tris-HCl, pH6.8 0.15g Bromophenol blue 8mL deionised water Add 2-mercapto-ethanol to 20% prior to use

129

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