Histone methyltransferase inhibitors: orally bioavailable, fast acting

Histone methyltransferase inhibitors: orally bioavailable, fast acting molecules with activity 1

against different human malaria species 2

3

Nicholas A. Malmquista,b

#, Sandeep Sundriyalc, Joachim Caron

c, Patty Chen

a,b, Benoit 4

Witkowskid, Didier Menard

d, Rossarin Suwanarusk

e, Laurent Renia

e, Francois Nosten

f,g, María 5

Belén Jiménez-Díazh, Iñigo Angulo-Barturen

h, María Santos Martínez

h, Santiago Ferrer

h, Laura 6

M. Sanzh, Francisco-Javier Gamo

h, Sergio Wittlin

i,j, Sandra Duffy

k, Vicky M. Avery

k, Andrea 7

Rueckerl, Michael J. Delves

l, Robert E. Sinden

l, Matthew J. Fuchter

c, Artur Scherf

a,b# 8

9

aUnité de Biologie des Interactions Hôte-Parasite, Institut Pasteur, Paris, France. 10

bCentre National de la Recherche Scientifique, Unité de Recherche Associée 2581, Paris, France. 11

cDepartment of Chemistry, Imperial College London, South Kensington Campus, London, 12

United Kingdom 13

dInstitut Pasteur du Cambodge, Malaria Molecular Epidemiology Unit, Phnom Penh, Cambodia 14

eSingapore Immunology Network (SIgN), Agency for Science, Technology and Research 15

(A*STAR), Biopolis, Singapore 16

fCentre for Tropical Medicine, Nuffield Department of Medicine, University of Oxford, Oxford, 17

United Kingdom 18

gShoklo Malaria Research Unit, Mahidol-Oxford Tropical Medicine Research Unit, Faculty of 19

Tropical Medicine, Mahidol University, Mae Sot, Thailand 20

hTres Cantos Medicines Development Campus, Diseases of the Developing World, 21

GlaxoSmithKline, Tres Cantos, Madrid, Spain 22

iParasite Chemotherapy Unit, Swiss Tropical and Public Health Institute, Basel, Switzerland 23

AAC Accepts, published online ahead of print on 24 November 2014Antimicrob. Agents Chemother. doi:10.1128/AAC.04419-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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jUniversity of Basel, Basel, Switzerland 24

kDiscovery Biology, Eskitis Institute, Griffith University, Nathan Campus, Brisbane, 25

Queensland, Australia 26

lDivision of Cell and Molecular Biology, Imperial College London, South Kensington, London, 27

United Kingdom 28

29

# Address correspondence to: [email protected] or [email protected]. 30

31

Running title: Antimalarial histone methyltransferase inhibitors32


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Abstract 33

Current antimalarials are under continuous threat due to the relentless development of drug 34

resistance by malaria parasites. We previously reported promising in vitro parasite killing 35

activity with the histone methyltransferase inhibitor BIX-01294 and its analogue TM2-115. Here 36

we further characterize these diaminoquinazolines for in vitro and in vivo efficacy and 37

pharmacokinetic properties to prioritize and direct compound development. BIX-01294 and 38

TM2-115 displayed potent in vitro activity with IC50 values <50 nM against drug sensitive 39

laboratory strains and multi-drug resistant field isolates including artemisinin refractory P. 40

falciparum isolates. Activity against ex vivo clinical isolates of both P. falciparum and P. vivax 41

were similar with potencies of 300-400 nM. Sexual stage gametocyte inhibition occurs at 42

micromolar levels, however, mature gametocyte progression to gamete formation is inhibited at 43

sub-micromolar concentrations. Parasite reduction ratio analysis confirms a fast asexual stage 44

rate of killing. Both compounds examined displayed oral efficacy in in vivo mouse models of P. 45

berghei and P. falciparum infection. The discovery of a rapid and broad-acting antimalarial 46

compound class targeting blood stage infection, including transmission stage parasites, and 47

effective against multiple malaria species reveals the diaminoquinazoline scaffold to be a very 48

promising lead for development into greatly needed novel therapies to control malaria. 49

50

Introduction 51

The continuous evolution of antimalarial drug resistance by Plasmodium parasites is a major 52

impediment to the elimination of this devastating disease. Artemisinin Combination Therapies 53

(ACTs) are the current mainstay of malaria chemotherapy, but the development of artemisinin 54

resistance in parasites has been reported in 2008-2009 along the Thai-Cambodian border (1). 55


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This underscores the need to validate new antimalarial targets within the parasite and develop 56

new antimalarial treatments based on novel scaffolds with desirable characteristics such as fast-57

killing activity against multiple parasite life stages, efficacy against multi-drug resistant strains 58

and multiple species of human malaria including P. vivax and favorable pharmacokinetics to 59

allow oral administration. 60

Epigenetic gene regulation mediated by histone modifying enzymes has been shown to play an 61

important role in malaria parasite transcriptional regulation, including the control of virulence 62

genes involved in immune evasion (2, 3). Histone lysine methyltransferase (HKMT) enzymes 63

present a novel potential target class for the development of antimalarials due to the association 64

of histone methylation at distinct lysine positions with both overall transcriptional activation 65

(H3K4me) and multi-copy gene family transcriptional repression (H3K9me) (4, 5). Indeed, half 66

of the identified P. falciparum HKMT enzymes were recently shown to be refractory to genetic 67

disruption (6). The essential and important regulatory role of HKMT enzymes on malaria 68

parasite biology, combined with information acquired through their increased attention in the 69

setting of cancer chemotherapy development (7), motivates the exploration of parasite HKMT 70

enzymes as a novel target class for antimalarial treatment. 71

We previously demonstrated BIX-01294, a histone methyltransferase inhibitor, and its analogue 72

TM2-115 (Fig. 1A) to cause rapid and irreversible parasite killing activity in vitro throughout the 73

intraerythrocytic asexual cycle (8). Importantly, the same class of molecule shows a ‘wake-up’ 74

effect on dormant malaria liver stage parasites (hypnozoites) suggesting an important role of 75

HKMT’s in this yet ill-defined biological liver stage (9). These promising antimalarial 76

characteristics led us to investigate these lead compounds against relevant multi-drug resistant 77

(including artemisinin resistance) field isolates and clinical isolates of P. falciparum and P. 78


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vivax. Activity against sexual stage gametocytes and their development into gametes, which are 79

responsible for malaria disease transmission, was also evaluated. To better assess the potential of 80

this novel class of molecules for preclinical development as antimalarials, we employed a 81

number of relevant tests such as oral bioavailability and efficacy in disease relevant animal 82

models. BIX-01294 and TM2-115 were subject to a Peters’ test in mice infected with P. berghei, 83

and a four-day test in SCID mice infected with P. falciparum parasites. Pharmacokinetic 84

analyses were undertaken to aid the interpretation of our in vivo results and inform further series 85

development. 86

87

Materials and Methods 88

Materials. Antimalarials including chloroquine and atovaquone were obtained from Sigma-89

Aldrich, artesunate was obtained from Sigma-Aldrich and Holly Pharmaceuticals Co. Ltd. DSM1 90

(10) was a gift from Akhil Vaidya (Drexel University College of Medicine). BIX-01294 and 91

TM2-115 were synthesized as previously described (11). Molecular masses of BIX-01294 and 92

TM2-115 free base compounds are both 491 g/mol, molecular masses of their trihydrochloride 93

salts are both 600 g/mol. 94

In vitro asexual stage parasite assays. Compound efficacy against drug-sensitive lab-strain 3D7 95

parasites and Cambodian artemisinin-resistant isolates were performed using a previously 96

described three day SYBR Green I growth and proliferation assay in a 96-well format (12). In 97

vitro parasite reduction ratio studies were performed as previously described (3). Cambodian 98

parasite isolates were collected in Palin province in 2010 and were culture-adapted at Institut 99

Pasteur in Cambodia, as previously described (13). All three strains harbor mutations (C580Y for 100

KH10-PL3 and KH10-PL10; R539T for 3601) in the propeller domain of the Kelch gene 101


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(PF3D7_1343700, K-13 propeller), recently associated with artemisinin resistance (14). Dose 102

response curves fitted and IC50 values calculated and compared using a one-way ANOVA in 103

GraphPad Prism Version 6.0e (San Diego, CA, USA). 104

Drug interaction studies. Isobologram analysis was performed using the fixed-ratio method as 105

described previously (15) in combination with the SYBR Green I assay to quantify P. falciparum 106

3D7 strain parasite growth and proliferation (12). BIX-01294 was analyzed in combination with 107

the P. falciparum dihydroorotate dehydrogenase inhibitor DSM1 (10) or the antimalarials 108

chloroquine (CQ), artesunate (AS) or atovaquone (ATQ). The fractional inhibitory concentration 109

(FIC) of each drug was calculated by dividing the IC50 for each drug in combination by the IC50 110

of each drug alone. Mean sum FIC values were computed to classify any interactions as 111

synergistic (≤0.5), antagonistic (≥4) or indifferent (0.5 < mean sum FIC < 4) (16). 112

Ex vivo clinical isolate parasite assays. Assays were performed as described previously (17) 113

with minor changes as follows. Six P. falciparum and four P. vivax isolates were collected from 114

malaria patients attending the clinics of Shoklo Malaria Research Unit (SMRU) Mae Sot region 115

of Tak Province, in Northwestern Thailand, under the following ethical guidelines in the 116

approved protocol OXTREC 027-025 (University of Oxford, Centre for Clinical Vaccinology 117

and Tropical Medicine, UK), from patients with no prior antimalarial therapy. After written 118

consent, blood samples were obtained by venipuncture in 5 ml volume lithium heparinized tubes. 119

Only samples with >90% of early ring stages were chosen for drug sensitivity testing after the 120

removal of leukocytes using a CF-11 column as described (18). Parasite susceptibility was tested 121

in parallel against chloroquine diphosphate (Sigma-Aldrich) and artesunate (Holly 122

Pharmaceuticals Co Ltd.) as previously described (17). Dose response curves and IC50 values 123


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using duplicate well data for each drug concentration were determined using ICESTIMATOR 124

(www.antimalarial-icestimator.net/MethodIntro.htm) (19, 20). 125

Gametocyte and dual gamete formation assays. Inhibition of early (Stage I-III) and late (Stage 126

IV-V) stage NF54 strain P. falciparum gametocyte viability was performed as previously 127

described (21). Male and female P. falciparum gamete formation assays were performed 128

essentially as described (22) for the “carry-over” format in which test compounds were present 129

for 24 hours with Stage V gametocytes and throughout gamete formation. For the “wash-out” 130

format, after 24 hours of compound incubation, gametocytes were washed three times over six 131

hours by total medium replacement and gamete formation was then triggered in the absence of 132

test compounds. 133

Ookinete formation assays. All work involving laboratory animals was performed in 134

accordance with the EU regulations 'EU Directive 86/609/EEC' and within the regulations of the 135

United Kingdom Animals (Scientific Procedures) Act 1986. The PbODA was set up identically 136

as described (23). Compounds were tested in quadruplicate independent biological replicates, 137

dose response curves fitted and IC50 values calculated using GraphPad Prism Version 6.0e (San 138

Diego, CA, USA). 139

In vitro cytotoxicity assays. Host cell cytotoxicity was determined in a 96-well format with a 140

starting HepG2 cell density of 10000 cells/well grown in DMEM (Life Technologies). Cells 141

were incubated with serial dilutions of test compounds for three days and resulting cell viability 142

was quantified using Promega CellTiterBlue. Cell viability as a function of test compound 143

concentration was fitted to survival curves to estimate compound IC50 values using GraphPad 144

Prism Version 6.0e (San Diego, CA, USA). 145


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Four-day test by Peters. In vivo efficacy against P. berghei was conducted as previously 146

described (24), with the modification that mice (n = 3-5) were infected with a GFP-transfected P. 147

berghei ANKA strain parasites (a gift from A. P. Waters and C. J. Janse, Leiden University, The 148

Netherlands), and parasitemia determined using standard flow cytometry techniques. 149

Compounds were dissolved or suspended in 70/30 Tween80/ethanol (vol/vol) and diluted 10-fold 150

in water before dosing. Experimental mice were treated at 4, 24, 48, and 72 hours post-infection 151

with a 50 mg/kg oral dose of the compound (4-day test by Peters) and were compared to an 152

infected control group for reduction in parasitemia on day 4 (96 hours post-infection) and for 153

mean survival (monitored up to 30 days post-infection). Five control mice and three treated mice 154

per treatment group were used. To support the interpretation of the in vivo efficacy results, 20 155

microliter plasma samples were collected from two of three mice from each treatment group for 156

mouse snapshot exposure studies. All animal studies were approved by the Veterinary Office of 157

Canton Basel-Stadt and animals were treated in accordance with institutional guidelines. 158

Four-day SCID mouse test. A cohort of age-matched female immunodeficient NOD-scid IL-159

2Rγcnull

mice (The Jackson Laboratory, Bar Harbor, ME) were engrafted with human 160

erythrocytes (generously provided by the Red Cross Transfusion Blood Bank in Madrid, Spain) 161

by daily injection with 1 ml of a 50% hematocrit erythrocyte suspension (RPMI 1640 medium, 162

25% (vol/vol) decomplemented human serum, 3.1 mM hypoxanthine) by intraperitoneal route 163

throughout the experiment as described (25). After seven days of injections mice reached 40% 164

human erythrocyte in peripheral blood and were intravenously infected with 2×107 P. falciparum 165

Pf3D70087/N9-infected erythrocytes (day 0). On day 3 after infection, mice were randomly 166

allocated to treatments that were administered once a day for 4 consecutive days by oral gavage 167


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at 10 ml/kg. Both BIX-01294 and TM2-115 were dissolved in 70% Tween-80/ 30% ethanol and 168

further diluted 1/10 in distilled water before administration. 169

Parasitemia was measured by flow cytometry in samples of peripheral blood stained with the 170

fluorescent nucleic acid dye SYTO-16 and anti-murine erythrocyte TER119 monoclonal 171

antibody (Pharmingen, San Diego, CA, USA) in serial 2 μL blood samples taken every 24 hours 172

until assay completion (26). 173

In the efficacy experiment the blood levels of BIX-01294 and TM2-115 in the mice were 174

measured in serial samples of peripheral blood (25 μl) taken by tail puncture at 0.25, 0.5, 1, 2, 4, 175

6, 8 and 23 hours after the first administration. The blood samples were immediately lysed by 176

mixing with 25 μl of water containing 0.1% saponin, frozen on dry ice and stored at -80ºC until 177

analysis. The compounds were extracted from 10 μL of each lysate by liquid-liquid extraction in 178

the MultiScreen Solvinert 0.45μm Hydrophobic PTFE 96- well plate system (Millipore) and 179

stored frozen at -80ºC until analysis by LC/MS/MS in AB Sciex API4000 (AB Sciex, 180

Framingham, MA). The compound concentration-versus-time data were analyzed by non-181

compartmental analysis (NCA) using WinNonlin® Professional Version 5.2 (Pharsight 182

Corporation, Mountain View, CA, USA). Additional statistical analysis was performed with 183

GraphPad Prism Version 5.01 (San Diego, CA, USA). 184

Efficacy was expressed as the daily exposure (AUC, μg·h/ml/day) of BIX-01294 and TM2-115 185

in whole blood necessary to reduce parasitemia at day 7 by 90 % with respect to vehicle-treated 186

mice (AUCED90). The AUCED90 was estimated by fitting a four parameter logistic equation for the 187

log10 [parasitemia at day 7 for individual i] versus the AUC0-23h of BIX-01294 and TM2-115 in 188

blood for individual i using GraphPad Prism 6.0e (San Diego, CA, USA). 189


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All the experiments were approved by the DDW Ethical Committee on Animal Research and 190

carried out in accordance with European Directive 2010/63/EU and the GSK Policy on the Care, 191

Welfare and Treatment of Animals. The animal studies were performed at DDW Laboratory 192

Animal Science facilities accredited by AAALAC. The human biological samples were sourced 193

ethically and their research use was in accord with the terms of the informed consents. 194

Functional hERG analysis. Functional hERG analysis was performed by Cyprotex 195

(Macclesfield, UK). Briefly, mammalian cells expressing the hERG potassium channel are 196

dispensed into 384-well planar arrays and hERG tail currents are measured by whole cell voltage 197

clamping. A range of concentrations (8 nM – 25 µM) of the test compound is then added to the 198

cells and a second recording of the hERG current is made. The percent change in hERG current 199

is then calculated. 200

201

Results 202

In vitro and ex vivo erythrocytic stage antimalarial activity. We previously reported that BIX-203

01294 and TM2-115 (Fig. 1A) possess similar antimalarial efficacy against both drug sensitive 204

and drug resistant laboratory strains of P. falciparum (8). The strains tested were resistant to 205

long-standing antimalarials for which resistance has developed in the field, namely chloroquine, 206

mefloquine and pyrimethamine. Reports of parasite resistance to the artemisinin derivatives, 207

which represent the cornerstone of modern antimalarial combination therapies, developing along 208

the Thai-Cambodian border prompted us to assess the efficacy of this compound series on 209

clinically relevant artemisinin-resistant field isolates (13, 27). Parasite growth and proliferation 210

in the presence of BIX-01294 and TM2-115 was assayed using three laboratory-adapted 211

artemisinin-resistant field isolates from Pailin, Cambodia. The three isolates (3601 PC, KH10-212


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PL03 and KH10-PL10) harbor mutations associated with artemisinin resistance and display a 213

resistant phenotype in a ring-stage survival assay (14.9%, 19.2% and 27.3% survival, 214

respectively, compared to 0.04% for sensitive 3D7 strain parasites) (14). The IC50 values for 215

either BIX-01294 or TM2-115 were not significantly different between artemisinin-resistant field 216

isolates and laboratory strain parasites (Table 1). These data suggest the mechanism of 217

artemisinin resistance (K13-propeller mutations) does not significantly impact the efficacy of 218

BIX-01294 and TM2-115, and suggests compounds developed from this series would be equally 219

effective against both artemisinin-sensitive and emerging artemisinin-refractory parasites. 220

Importantly, the IC50 values of BIX-01294 and TM2-115 are >100-fold higher against HepG2 221

human hepatic carcinoma cells (Table 1) as determined in a similar assay, indicating selectivity 222

for parasites over host cells (Fig. 1B). 223

Of the Plasmodium species that infect humans, P. falciparum is the major contributor to 224

worldwide malaria mortality. Though responsible for fewer malaria fatalities, P. vivax is the 225

main cause of global morbidity and responsible for instances of malaria relapse due to the ability 226

of this species to remain as dormant hypnozoites for years within the liver of a previously 227

infected individual (28). To identify whether BIX-01294 and TM2-115 have cross-species 228

antimalarial activity, we assayed these compounds against clinical isolates of P. falciparum and 229

P. vivax using an ex vivo red blood cell stage parasite progression assay (17). The data revealed 230

similar IC50 values against either P. falciparum or P. vivax clinical isolates for both BIX-01294 231

and TM2-115 (Table 2). The overall IC50 values in the ex vivo assay are higher than those 232

observed in the in vitro assay, including for chloroquine, likely due to experimental differences 233

between the ex vivo and in vitro assays. Notably, the ex vivo assay reports on the progression 234

from early to late stage parasites, and thus reveals the effect of compound exposure during a 235


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single cycle of the 48-hour erythrocytic stage. These data suggest compounds developed from 236

this series would be equally effective against erythrocytic stages of the two most clinically 237

relevant species of human malaria parasites and are able to exert their killing effect on the time 238

scale of a single 48-hour erythrocytic replication cycle. 239

Parasite reduction ratio (PRR) analysis. Currently used antimalarials display a wide range of 240

parasite killing rates (3). The inherent killing rate of individual antimalarials impacts not only the 241

therapeutic onset and thereby the efficacy of a given treatment, but is also a major consideration 242

in choosing combination therapy partners for novel antimalarial formulations. To best determine 243

the rate of parasite killing by BIX-01294 and TM2-115, we performed a parasite reduction ratio 244

(PRR) analysis that quantifies the number of viable parasites after exposure to 10-fold the IC50 245

concentrations of test antimalarials for various lengths of time (3). P. falciparum parasites (strain 246

3D7A) were exposed to BIX-01294 or TM2-115 for periods ranging from 24 to 120 hours or 24 247

to 48 hours, respectively, after which time the compounds were washed out. Treated parasites 248

were serially diluted into 96-well culture plates to determine the number of viable parasites 249

remaining after 21-28 days of culture with fresh erythrocytes. Results from this analysis show 250

that both BIX-01294 and TM2-115 display a rapid killing profile without any lag phase, 251

essentially eliminating all viable parasites after a 48-hour exposure to either compound (Fig. 2). 252

For BIX-01294, the log PRR value, representing the logarithm of the number of parasites 253

eliminated in one erythrocytic cycle, was 4.7, and the PCT99.9% value, representing the time to 254

eliminate 99.9% of the starting parasitemia, was 29 hours. TM2-115 displayed very similar 255

effects in an abbreviated assay for which log PRR and PCT99.9% were not calculated. Results 256

from this PRR analysis reveal BIX-01294 and TM2-115 produce a rapid killing effect slightly 257

faster than chloroquine, the second most rapidly acting antimalarial after artemisinin (3). 258


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In vitro drug interaction studies. We performed fixed-ratio isobologram analyses to determine 259

whether any apparent interaction exists in vitro between BIX-01294 and existing compounds 260

with known antimalarial mechanisms of action (15, 29). Mean sum Fractional Inhibitory 261

Concentration (FIC) values of 1.1-1.4 for each drug combination reveal little if any interaction 262

between BIX-01294 and the other parasite killing compounds tested (Fig. 3). These data suggest 263

that compounds developed from this series would be effective if employed in combination with 264

existing antimalarials without a concern for drug antagonism. 265

In vitro transmission stage activity. Sexual stage gametocytes are responsible for the 266

transmission of malaria parasites from the infected host to the mosquito vector and then 267

subsequent additional human hosts. Of the antimalarials currently in world-wide use, only 268

primaquine is proven effective at reducing the infectivity of the generally drug-insensitive 269

mature gametocyte (30). Unfortunately, contraindications for primaquine use, including glucose 270

6-phosphate 1 dehydrogenase (G6PD) deficiency, which is prevalent in malaria-endemic areas, 271

prompts the search for new compounds with efficacy against gametocytes. To examine whether 272

BIX-01294 or TM2-115 possess potential transmission-blocking activity, we determined IC50 273

values for each compound against P. falciparum gametocytes in either the early (Stage I-III) or 274

late (Stage IV-V) phases of the 10-14 day gametocyte developmental period. The data show IC50 275

values of 1-4 µM for BIX-01294 or TM2-115 against either early- or late-stage gametocytes 276

using a high-content imaging based assay (Table 3). 277

We also investigated the effect of BIX-01294 and TM2-115 on mature Stage V gametocyte 278

development into male and female gametes (22). The assay employed reports on the ability of a 279

test compound to not necessarily kill mature Stage V gametocytes directly, but rather to block 280

their functional viability and prevent the onward development necessary for parasite 281


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transmission to the mosquito. Assays were performed to identify effects on gametocytes prior to 282

gamete formation in a “wash-out” format and on gametocytes during gamete formation in a 283

“carry-over” format. Results from these dual gamete formation assays show BIX-01294 and 284

TM2-115 to have inhibitory effects on both male and female gamete formation in both the wash-285

out and carry-over experiments (Table 3). Male gametes are particularly affected, with IC50 286

values of 20-140 nM for either compound in both assay formats. These results suggest treatment 287

with either compound irreversibly compromises the capacity of Stage V gametocytes to develop 288

into gametes and strongly suggests transmission-blocking activity via decreasing the number of 289

viable male gametes. 290

Additionally, we examined the effect of BIX-01294 and TM2-115 on P. berghei ookinete 291

development, the parasite stage resulting from the zygote formed after gamete fertilization. This 292

assay reports on the ability of a drug to inhibit the transformation of ex vivo P. berghei mature 293

gametocytes into ookinetes, encompassing gamete formation, fertilization and zygote 294

development into the mature ookinete. Data from these experiments show both test compounds 295

inhibit ookinete development with IC50 values of 6-7 µM (Table 3). 296

In vivo erythrocytic stage activity against P. berghei in a mouse model. We previously 297

demonstrated our diaminoquinazoline compounds possess activity against malaria parasites in an 298

animal model of infection after intraperitoneal administration (8). To explore the oral efficacy of 299

BIX-01294 and TM2-115 we performed a four-day Peters’ test, which examines the ability of 300

compounds to reduce or clear blood stage infection (31). Mice were infected with P. berghei 301

parasites and then treated via oral delivery at 4, 24, 48 and 72 hours post-infection with 50 mg/kg 302

of free-base or salt formulations of BIX-01294 or TM2-115 (Fig. 4A). Treatment with BIX-303

01294 free-base or salt formulations yielded a 99.9% reduction in parasitemia versus untreated 304


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controls on day four post-infection (Fig. 4B). Treatment with TM2-115 free-base or salt 305

formulations produced a 95% or 87% reduction in parasitemia versus untreated controls, 306

respectively, on day four post-infection (Fig. 4B). These reductions in parasitemia were reflected 307

in the overall survival rates of treated mice, where BIX-01294 treated mice survived to 12-16 308

days post-infection while TM2-115 treated mice survived 6-10 days post-infection (Fig. 4C). 309

Pharmacokinetic analysis revealed a long serum half-life (t1/2) for both free-base and salt 310

formulations of BIX-01294, though the salt form yields higher serum levels relative to the base 311

form, but a much shorter t1/2 for either form of TM2-115 (Fig. 4D). Thus, increased exposure to 312

BIX-01294 relative to TM2-115 may account for the difference in efficacy between the two 313

compounds. Though neither compound treatment resulted in complete parasite clearance, these 314

data show this diaminoquinazoline series exhibits oral efficacy against P. berghei rodent malaria 315

parasites in a mouse model of infection. 316

In vivo erythrocytic stage activity against P. falciparum in a humanized mouse model. To 317

obtain a proof of concept of the therapeutic efficacy of BIX-01294 and TM2-115 against P. 318

falciparum human malaria parasites in vivo, we employed NODscidIL2Rγnull

mice engrafted 319

with human erythrocytes. Mice infected with P. falciparum were treated by oral delivery at days 320

3, 4, 5 and 6 post-infection with 75 or 100 mg/kg and parasite levels were measured on days 3-7 321

(Fig. 5A). The levels of BIX-01294 and TM2-115 in blood were also measured for 23 h after the 322

first dose in the same animals (Fig. 5B). In mice treated with BIX-01294 parasitemia fell below 323

the limit of detection at day six or five of the experiment and, consistently, most parasites in 324

blood were pyknotic after 48 h of exposure (Fig. 5C). In infected mice treated with TM2-115 the 325

parasite load remained essentially at the same level as when the infected mice were first treated 326

and parasites in blood were a mixture of pyknotic and replicating parasites after 48 h of exposure 327


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(Fig. 5C). The difference in therapeutic response between BIX-01294 and TM2-115 is consistent 328

with the 5-fold higher exposure of the former mice (BIX-01294 dose normalized AUC 0.25 and 329

0.31 µg·h/ml per mg/kg versus TM2-115 dose normalized AUC of 0.07 and 0.06 µg·h/ml per 330

mg/kg at 75 and 100 mg/kg, respectively) rather than any intrinsic difference in potency or 331

activity in vivo. Together, these data show that our diaminoquinazoline series are orally 332

efficacious and kill P. falciparum rapidly in vivo. 333

Functional hERG analysis. Quinazoline-containing molecules have been known to inhibit the 334

human cardiac voltage-gated potassium channel Ether-a-go-go Related Gene (hERG), presenting 335

a potential liability for further series development (32). To assess whether our two 336

diaminoquinazoline compounds possess any inhibitory activity against hERG, we obtained 337

electrophysiology-based functional inhibition data for these two compounds (Fig. 6). BIX-01294 338

began inhibiting hERG channel signal at concentrations above 10 µM, producing 65% inhibition 339

at 25 µM, the highest concentration investigated. TM2-115 displayed very little hERG channel 340

inhibition across the concentration range tested, inhibiting 17% at 25 µM. These results indicate 341

BIX-01294 inhibits hERG at concentrations higher than those found to produce overall toxicity 342

to HepG2 cells and greater than 500-fold higher than the in vitro IC50 against parasites. 343

344

Discussion 345

The identification of histone post-translational modifications playing a key role in transcriptional 346

regulation in the malaria parasite P. falciparum led us to undertake a chemical biology approach 347

to investigate and manipulate this important mechanism of parasite biology (4, 33). Histone 348

methylation in particular has been linked to both general transcriptional activity and activation 349

and silencing of multi-copy gene families that are implicated in immune evasion and infected red 350


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blood cell sequestration (5, 6, 34). We initially screened several known histone methyltransferase 351

inhibitors against parasites in culture and discovered that the diaminoquinazoline BIX-01294 and 352

related analogues reduced parasite histone methylation levels and showed promising preliminary 353

antimalarial characteristics against blood stage parasites (8). Subsequent demonstration of 354

activity on liver stage hypnozoites (9) further increased interest in these molecules for their 355

potential in treating different life cycle stages of human malaria parasites. The current studies 356

were designed to further investigate this series and provide data to guide development of 357

diaminoquinazolines as potential novel antimalarials. 358

BIX-01294 and TM2-115 show comparable in vitro efficacy against drug-sensitive laboratory 359

parasites and artemisinin-resistant field isolates. Artemisinin-based combination therapies 360

(ACTs) represent the front-line therapy in many malaria endemic areas. Emerging resistance to 361

artemisinin and partner drugs in Southeast Asia threatens the effectiveness of all ACTs (1, 35-362

38). Artemisinin resistance manifests as a reduced effectiveness of artemisinins against ring-363

stage parasites (14). Compounds with efficacy against parasites resistant to this integral 364

component of antimalarial combination therapy will be important when developing next-365

generation therapies with worldwide efficacy. Importantly, we have demonstrated BIX-01294 366

and TM2-115 to be equally effective throughout the erythrocytic cycle, including against ring-367

stage parasites (8). This indicates that our diaminoquinazolines represent a promising compound 368

class with a high likelihood of activity against existing and emerging multi-drug resistant 369

parasites strains. 370

Activity against both P. falciparum and P. vivax clinical isolates demonstrates cross-species 371

activity toward the two most relevant human malaria pathogens for this compound series. This 372

cross-species activity may be due to the high conservation of HKMT proteins in P. falciparum 373


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and P. vivax and their assumed conserved role in transcriptional regulation in all Plasmodium 374

species. Activity against several Plasmodium species that affect humans is a very desirable 375

characteristic of any potential new antimalarial therapy. 376

We previously showed a rapid parasite killing effect in treatment and wash-out experiments (8). 377

A more robust analysis of parasite killing rate has since been developed and this improved 378

method was employed to better characterize the rate and extent of parasite killing by BIX-01294 379

and TM2-115 (3). These studies revealed parasite-killing rates by diaminoquinazoline 380

compounds to be slightly more rapid than chloroquine, the fastest parasite killer currently in 381

clinical use after artemisinin derivatives. An advantage of this compound series is its 382

demonstrated killing activity during the entire asexual blood stage development (8). This is 383

probably due to the need for continual activation of genes throughout the 48-hour blood stage 384

development, a process linked to epigenetic transcriptional regulation via histone methylation. 385

Rapid killing antimalarials are important for reducing initial parasite loads, especially in severe 386

malaria cases, leading to an improved clinical response, and reducing overall parasite numbers 387

lowering the potential for resistance development to both that drug and any partner drug of a 388

combination chemotherapy. Importantly, the speed of action of an antimalarial is directly related 389

to its mechanism of action. Thus, future compounds from this series that maintain activity 390

against the same target will retain this rapid killing effect. 391

Drug interaction studies in vitro showed BIX-01294 to have no significant interaction when 392

tested in combination with chloroquine, artesunate, atovaquone or the parasite dihydroorotate 393

dehydrogenase inhibitor DSM1. The overall parasite killing profile, together with a novel 394

chemical structure and activity against characterized resistant strains, may indicate BIX-01294 395

targets a parasite mechanism distinct from these other molecules with known mechanisms of 396


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action. The lack of any antagonism indicates BIX-01294 would in theory be a suitable partner 397

drug in any combination antimalarial therapy comprised of compounds tested in these studies. As 398

we develop this compound series further, interactions with potential partner drugs could be 399

similarly investigated. 400

Populations of gametocytes, the parasite form responsible for malaria transmission, are female 401

dominated with a natural ratio of females-to-males of approximately 4:1 (22). Compound 402

activity against developing mixed-sex gametocytes appeared to be low relative to asexual blood 403

stage parasites. Mean inhibitory concentrations against both early- and late-stage gametocyte 404

viability were above one micromolar, comparable to that against human HepG2 cells, which we 405

use as a measure of potential host cell toxicity. Inhibitory activity against subsequent mosquito 406

stage ookinete development of P. berghei occurred at similarly high compound concentrations. 407

However in P. falciparum, dual gamete formation experiments demonstrated inhibition of male 408

gamete formation in treated and washed mature Stage V gametocytes at 20-140 nM, revealing 409

potent and irreversible inhibition of the functional viability of gametocytes to progress to 410

gametes. Overall, male gametocytes/gametes were 10-fold more susceptible to inhibition than 411

the more abundant female gametes, and TM2-115 was roughly 5-fold more potent than BIX-412

01294. As such, while these diaminoquinazoline compounds appear less effective at inhibiting 413

gametocyte and ookinete development and maturation, they are indeed effective at inhibiting 414

gametocyte onward functional viability to develop into gametes – an essential first step in 415

establishing an infection in the insect vector. The current understanding of transcriptional 416

regulation in sexual stage parasites is very limited. Differential expression profiles of the ten 417

annotated methyltransferase enzymes in asexual stage parasites compared to sexual stage 418


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gametocytes is apparent (plasmodb.org) and may account for differential compound activities in 419

these different life cycle stages. 420

In vivo oral efficacy studies were performed in both mice harboring P. berghei rodent malaria 421

and humanized SCID mice harboring P. falciparum malaria. Against P. berghei, BIX-01294 and 422

TM2-115 were effective in decreasing parasite levels 99% to 87%, respectively, at day four post-423

infection after four oral doses of 50 mg/kg. Both the free-base and salt form of each compound 424

was similarly effective. Mouse survival was extended relative to the ability of each compound to 425

decrease overall parasite levels, but neither BIX-01294 nor TM2-115 treatment led to a cure in 426

these experiments. These results are similar to previous onset of activity and recrudescence 427

studies where BIX-01294 or TM2-115 was administered via intraperitoneal injection to patently 428

infected mice (8). In that study, parasite levels decreased with compound treatment, but neither 429

treatment resulted in a cure. Related pharmacokinetic analysis revealed a plasma half-life of >24 430

hours for BIX-01294, whereas TM2-115 was rapidly cleared from plasma. This pharmacokinetic 431

differential, leading to different compound exposure levels, is likely to explain the difference in 432

efficacy between the two molecules tested with regards to decreased P. berghei levels and 433

survival outcome. In a mouse model of P. falciparum infection, BIX-01294 and TM2-115 434

performed similarly as in the P. berghei models. Four oral doses of TM2-115 beginning on day 435

three post-infection decreased parasite levels relative to vehicle controls, though overall parasite 436

levels remained essentially steady from the onset of treatment. Similar treatment of patent P. 437

falciparum infection with BIX-01294 in the SCID mouse model produced a much greater 438

decrease in parasite numbers, to below the limit of detection on day five or six post infection, i.e. 439

after three or four doses of 100 or 75 mg/kg, respectively. Together, these in vivo efficacy studies 440

show this compound series to be orally bioavailable and active against both P. berghei, the 441


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parasite species of standard mouse models of malaria infection, and P. falciparum, the most 442

relevant human malaria parasite. These studies both establish oral efficacy of this compound 443

series and might support the use of P. berghei models, which are readily available relative to the 444

P. falciparum model, to facilitate future compound progression. Importantly, the humanized 445

SCID mouse model indicates that diaminoquinazoline compounds kill P. falciparum rapidly in 446

vivo in a range of concentrations in blood that might be achievable in humans. 447

In summary, our in vitro and in vivo antimalarial activity studies of this diaminoquinazoline 448

compound series indicate very promising activity against sexual and multiple asexual stage 449

Plasmodium species with various genetic backgrounds including multidrug-resistant profiles. 450

These results support further development of this compound series as a novel antimalarial class, 451

which is currently underway with regards to red cell stage efficacy and specificity and compound 452

stability (39). Through iterative medicinal chemistry efforts, we aim to improve the 453

pharmacokinetic characteristics of next-generation molecules while retaining the rapid killing 454

profile and improving efficacy and specificity against P. falciparum and P. vivax blood-stage 455

parasites and gametocyte functional viability. Ongoing target identification and subsequent target 456

characterization efforts will greatly aid the progression of this compound series. 457

458

Acknowledgments 459

The authors wish to thank Xavier Ding and Brice Campo of Medicines for Malaria Venture for 460

helpful discussions and aid in coordinating some of the collaborative efforts. We thank Jolanda 461

Kamber for assistance in performing the P. berghei in vivo assay and Christoph Siethoff (Swiss 462

BioQuant) for the determination of compound levels in P. berghei infected mice. We are also 463

grateful to the Therapeutic Efficacy, Pharmacology and Laboratory Animal Science groups at 464


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GSK Tres Cantos Medicines Development Campus for assistance. We thank L. D. Shultz and 465

The Jackson Laboratory for providing access to nonobese diabetic scid IL2Rgc null mice 466

through their collaboration with GSK Tres Cantos Medicines Development Campus. We are 467

indebted to Susan A. Charman of Monash University for critically reviewing the manuscript. 468

S.S. acknowledges the European Commission for a Marie Curie International Incoming 469

Fellowship (Agreement No. 299857). L.R. was supported by funding from the Singapore 470

Immunology Network (SIgN) and the Horizontal Programme on Infectious Diseases under the 471

Agency for Science, Technology and Research (A*STAR, Singapore). Shoklo Malaria Research 472

Unit is part of the Mahidol Oxford University Research Unit, supported by the Wellcome Trust 473

of Great Britain. V.A. acknowledges support from the Australian Research Council 474

(LP120200557). A.S. acknowledges support from the European Research Council. 475

476

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631

632


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Figure legends: 633

634

FIG. 1. Structures of BIX-01294 and TM2-115 and compound specificity. (A) Compound 635

structures. (B) Cell growth and proliferation of P. falciparum 3D7 strain parasites (solid 636

symbols) or HepG2 cells (open symbols) in respective three-day assays in the presence of BIX-637

01294 (squares) or TM2-115 (triangles). Data are mean ± SD of duplicate measurements from a 638

single representative experiment. 639

640

FIG. 2. Parasite reduction ratio analysis. P. falciparum 3D7A strain parasites were exposed to 641

10x IC50 concentrations of BIX-01294 (open squares), or TM2-115 (open triangles), chloroquine 642

(open circles) or artemisinin (open inverted triangles) for the durations indicated, then compound 643

was washed out and parasites were diluted to determine the number of viable parasites 644

remaining. Values represent mean ± SD of quadruplicate parasite measurements. 645

646

FIG. 3. Isobologram analysis of BIX-01294 and various antimalarials. Fixed-ratio isobolograms 647

were constructed from growth and proliferation assays using fixed ratios of BIX-01294 with 648

other antimalarial compounds. Fractional inhibitory concentrations (FIC) are based on IC50 649

values of combination treatment relative to BIX-01294 or other antimalarial alone. FIC data 650

represent mean ± SD of two or three independent experiments. BIX, BIX-01294; CQ, 651

chloroquine; AS, artesunate; ATQ, atovaquone; DSM1, (5-methyl[1,2,4]triazolo[1,5-652

a]pyrimidin-7-yl)naphthalen-2-ylamine. 653

654


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FIG. 4. Peters test for oral efficacy in a mouse model of P. berghei malaria infection and 655

corresponding pharmacokinetics. (A) Mice were infected with P. berghei parasites at time zero 656

and treated with 50 mg/kg of compound at 4, 24, 48 and 72 hours post-infection. (B) Parasite 657

burden was measured at day four post-infection. (C) Mouse survival was followed thereafter. 658

The control group (open circles) contained five mice, the BIX-01294 (squares) and TM2-115 659

(triangles) treatment groups contained three mice. Data in (B) are mean ± SD and all replicates 660

are shown. (D) Pharmacokinetic properties of compounds delivered via oral dosing. Plasma 661

levels of compounds were determined 1, 4 and 24 hours after oral delivery of 50 mg/kg of each 662

compound in free base (filled symbols) or salt form (open symbols). 663

664

FIG. 5. Four-day test for oral efficacy in a humanized mouse model of P. falciparum infection. 665

(A) Mice were infected with P. falciparum and treated on days 3, 4, 5 and 6 post-infection with 666

either 75 or 100 mg/kg of BIX-01294 (open or closed squares) or TM2-115 (open or closed 667

triangles). Parasitemia was quantified on days three to six post-infection. Each symbol represents 668

an individual mouse except for vehicle-treated controls (black circles, n = 2 mice). (B) The 669

concentration of BIX-01294 (open or closed squares) and TM2-115 (open or closed triangles) 670

was monitored in the blood of each individual mouse of the efficacy study during the first 23 h 671

after the first oral dose administration. Each symbol represents an individual mouse. (C) The 672

effect of BIX-01294 or TM2-115 on parasites in peripheral blood of mice was assessed by 673

microscopy 48 h after the start of treatment. 674

675

FIG. 6. Functional hERG analysis. Compounds BIX-01294 (open squares), TM2-115 (open 676

triangles) and positive control quinidine (open circles) were tested for inhibition of the hERG 677


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potassium channel. Mean measurements ± SD are presented and the data are fitted to a standard 678

inhibition curve. 679

680


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Tables: 681

P. falciparum

strain IC50

(nM)

P. falciparum

ArtR isolate IC50 (nM)

compound

treatment 3D7 3601 PC KH10-PL03 KH10-PL10

HepG2

IC50 (nM)

BIX-01294 19 ± 3 35 ± 14 30 ± 13 35 ± 16 4850 ±

1090

TM2-115 32 ± 5 48 ± 18 37 ± 13 46 ± 24 4690 ±

1180

dihydroartemisinin 0.63 ± 0.41 1.3 (1.0-1.6) 0.32 (0.24-0.44) 0.32 (0.27-0.37) -

artesunate 0.97 ± 0.94 1.5 (1.1-2.1) 0.82 (0.68-0.99) 0.74 (0.54-1.0) -

chloroquine 8.4 ± 1.2 68 ± 25

50 ± 6 45 ± 9 -

682

TABLE 1. In vitro compound efficacy against a drug sensitive (3D7) laboratory strain and multi-683

drug resistant including artemisinin-resistant (ArtR) field isolates from Pailin, Cambodia. IC50 684

values were determined in a three-day SYBR Green I based growth and proliferation assay. 685

HepG2 cell viability was determined in a three-day CellTiterBlue assay. Values are mean ± SD 686

of two or three experiments for BIX-01294, TM2-115 and chloroquine. For dihydroartemisinin 687

and artesunate, values are mean ± SD from 20-21 experiments for 3D7 strain parasites or mean 688

and 95% CI for a single IC50 determination for the ArtR strains. 689

690


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compound

treatment

P. falciparum

IC50 (nM)

P. vivax

IC50 (nM)

BIX-01294 280 ± 90 390 ± 90

TM2-115 340 ± 160 240 ± 70

chloroquine 66 ± 37 51 ± 21

artesunate 1.1 ± 0.5 0.9 ± 0.4

692

TABLE 2. Ex vivo compound efficacy against clinical isolates of P. falciparum and P. vivax. 693

IC50 values were determined in a parasite maturation assay based on microscopic evaluation of 694

parasite progression from young ring-stage to mature schizont-stage parasites. Values are mean ± 695

SD of duplicate measurements of four to seven isolates per parasite species. 696

697

P. falciparum

gametocyte viability

P. falciparum gamete formation P.

berghei

ookinete

viability

(M)

male female

compound

early-

stage

(M)

late-

stage

(M)

carry-

over

(M)

wash-

out

(M)

carry-

over

(M)

wash-

out

(M)

BIX-01294 1.07 ±

0.08

3.84 ±

0.36

0.120 ±

0.050

0.142 ±

0.014

0.727 ±

0.150

1.38 ±

0.26 8.4 ± 1.5

TM2-115 1.30 ±

0.08

2.63 ±

0.25

0.022 ±

0.015

0.022 ±

0.004

0.203 ±

0.020

0.264 ±

0.017 7.9 ± 1.7

artesunate 0.0047 ±

0.0003

0.0076 ±

0.0004 - - - - -

698

TABLE 3. In vitro compound efficacy against early- and late-stage gametocyte viability, gamete 699

formation and ookinete viability. Values represent the mean ± SEM of the fitted data for two to 700

six independent IC50 determinations. 701


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-9 -8 -7 -6 -50

20

40

60

80

100

120

log [compound] (M)

% c

ell

gro

wth

& p

rolif

era

tio

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BIX-01294 vs 3D7

TM2-115 vs 3D7

BIX-01294 vs HepG2

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(A)

(B)


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0 24 48 72 96 1200

1

2

3

4

5

6

treatment time (hours)

log

(v

iab

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ara

site

s +

1) BIX-01294

TM2-115

chloroquine

artemisinin


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infect

48 72 96 (hours) 24 0 dose dose dose dose

parasitemia

survival

0 4 8 12 160

20

40

60

80

100

time (days)

% s

urv

iva

l

0

20

40

60

80

treatment compound

% p

ara

site

mia

0 4 8 12 16 20 240

20

40

60

80

time (hours)

pla

sm

a c

on

c. (

ng

/ml)

untreated

BIX-1294 base

BIX-1294 salt

TM2-115 base

TM2-115 salt

(A) (B)

(C) (D) on February 6, 2018 by guest

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Documents

Histone methyltransferase inhibitors: orally bioavailable, fast acting