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
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691
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
n
BIX-01294 vs 3D7
TM2-115 vs 3D7
BIX-01294 vs HepG2
TM2-115 vs HepG2
N
NMeO
MeO
NH
N
N Me
N
N
NO
MeO
NH
N
N Me
NMe
!"#$%&'()* +,'$&&-*
(A)
(B)
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0 24 48 72 96 1200
1
2
3
4
5
6
treatment time (hours)
log
(v
iab
le p
ara
site
s +
1) BIX-01294
TM2-115
chloroquine
artemisinin
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4
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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