A novel multiple-stage antimalarial agent that inhibits protein synthesis

Article abstract of DOI:10.1038/nature14451

There is an urgent need for new drugs to treat malaria, with broad therapeutic potential and novel modes of action, to widen the scope of treatment and to overcome emerging drug resistance. Here we describe the discovery of DDD107498, a compound with a po

Full text of DOI:10.1038/nature14451

ARTICLE
doi:10.1038/nature14451
A novel multiple-stage antimalarial agent
that inhibits protein synthesis
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Beatriz Baraga n˜ a , Irene Hallyburton , Marcus C. S. Lee {, Neil R. Norcross , Raffaella Grimaldi , Thomas D. Otto ,
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William R. Proto , Andrew M. Blagborough , Stephan Meister , Grennady Wirjanata , Andrea Ruecker , Leanna M. Upton ,
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Tara S. Abraham , Mariana J. Almeida , Anupam Pradhan , Achim Porzelle , Mar ´ı a Santos Mart ´ı nez , Judith M. Bolscher ,
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Andrew Woodland , Suzanne Norval , Fabio Zuccotto , John Thomas , Frederick Simeons , Laste Stojanovski ,
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Maria Osuna-Cabello , Paddy M. Brock , Tom S. Churcher , Katarzyna A. Sala , Sara E. Zakutansky , Mar ´ı a Bel e´ n Jim e´ nez-D ´ı az ,
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Laura Maria Sanz , Jennifer Riley , Rajshekhar Basak , Michael Campbell , Vicky M. Avery , Robert W. Sauerwein ,
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Koen J. Dechering , Rintis Noviyanti , Brice Campo , Julie A. Frearson , I n˜ igo Angulo-Barturen , Santiago Ferrer-Bazaga ,
Francisco Javier Gamo , Paul G. Wyatt , Didier Leroy , Peter Siegl , Michael J. Delves , Dennis E. Kyle , Sergio Wittlin ,
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Jutta Marfurt , RicN. Price , Robert E. Sinden , ElizabethA. Winzeler , Susan A. Charman , LidiyaBebrevska , David W. Gray ,
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Simon Campbell , Alan H. Fairlamb , Paul A. Willis , Julian C. Rayner , David A. Fidock , Kevin D. Read & Ian H. Gilbert
There is an urgent need for new drugs to treat malaria, with broad therapeutic potential and novel modes of action, to
widen the scope of treatment and to overcome emerging drug resistance. Here we describe the discovery of DDD107498,
a compound with a potent and novel spectrum of antimalarial activity against multiple life-cycle stages of the
Plasmodium parasite, with good pharmacokinetic properties and an acceptable safety profile. DDD107498 demon-
strates potential to address a variety of clinical needs, including single-dose treatment, transmission blocking and
chemoprotection. DDD107498 was developed from a screening programme against blood-stage malaria parasites; its
molecular target has been identified as translation elongation factor 2 (eEF2), which is responsible for the
GTP-dependent translocation of the ribosome along messenger RNA, and is essential for protein synthesis. This
discovery of eEF2 as a viable antimalarial drug target opens up new possibilities for drug discovery.
The World Health Organization estimates that there were approxi- the parasites, but suffered from poor physicochemical properties.
mately 200 million clinical cases and 584,000 deaths from malaria in Chemical optimization (Fig. 1 and Extended Data Fig. 1) led to
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013, predominantly among children and pregnant women in sub- DDD107498 with improved physicochemical properties (Tables 1
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Saharan Africa . The malaria parasite has developed resistance to and 2 in Supplementary Methods) and a 100-fold increase in
many of the current drugs, including emerging resistance to the core potency. The key stages involved were replacing the bromine with a
artemisinin component of artemisinin-based combination therapies fluorine atom to reduce molecular mass and lipophilicity; replacing
that constitute current first-line therapies . To support the current the 3-pyridyl substituent with an ethyl-pyrrolidine group; and addi-
treatment and eradication agenda , there are several requirements for tion of a morpholine group via a methylene spacer. Initial cost-of-
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3
new antimalarials: novel modes of action with no cross-resistance to goods estimates and likely human dose projections suggest a low cost
current drugs; single-dose cures; activity against both the asexual (approximately US$1 per treatment), which is important, given that
blood stages that cause disease and the gametocytes responsible for most of the patient population is living in poverty.
transmission; compounds that prevent infection (chemoprotective
agents); and compounds that clear Plasmodium vivax hypnozoites Blood-stage activity and developability
4
from the liver (anti-relapse agents) .
DDD107498 showed excellent activity against 3D7 parasites: 50%
effective inhibitory concentration (EC ) 5 1.0 nM (95% confidence
interval (CI) 0.8–1.2 nM); EC90 5 2.4 nM (95% CI 2.0–2.9 nM);
EC99 5 5.9 nM (95% CI 4.5–7.6 nM), (n 5 39). It was also almost
50
Discovery of a novel antimalarial
A phenotypic screen of the Dundee protein kinase scaffold library
5
(
then 4,731 compounds) was performed against the blood stage of the equally active against several drug-resistant strains (Extended Data
6
multi-drug-sensitive Plasmodium falciparum 3D7 strain. A com- Fig. 2a) . Furthermore, DDD107498 was more potent than artesunate
pound series from this screen, based on a 2,6-disubstituted quin- in ex vivo assays against a range of clinical isolates of both P. falci-
oline-4-carboxamide scaffold, had sub-micromolar potency against parum (median EC50 5 0.81 nM (range 0.29–3.29 nM), n 5 44) and
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Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK. Department of Microbiology and Immunology, Columbia
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University College of Physicians and Surgeons, New York, New York 10032, USA. Malaria Programme, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge CB10 1SA, UK.
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Department of Life Sciences, Imperial College, London SW7 2AZ, UK. University of California, San Diego, School of Medicine, 9500 Gilman Drive 0760, La Jolla, California 92093, USA. Global Health
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and Tropical Medicine Division, Menzies School of Health Research, Charles Darwin University, PO Box 41096, Casuarina, Darwin, Northern Territory 0811, Australia. Department of Global Health,
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College of Public Health University of South Florida, 3720 Spectrum Boulevard, Suite 304, Tampa, Florida 33612, USA. GlaxoSmithKline, Tres Cantos Medicines Development Campus-Diseases of the
Developing World, Severo Ochoa 2, Tres Cantos 28760, Madrid, Spain. TropIQ Health Sciences, Geert Grooteplein 28, Huispost 268, 6525 GA Nijmegen, The Netherlands. Centre for Drug Candidate
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Optimisation, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia. Eskitis Institute, Brisbane Innovation Park, Nathan Campus, Griffith University, Queensland 4111, Australia.
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Malaria Pathogenesis Laboratory, Eijkman Institute for Molecular Biology, Jalan Diponegoro 69, 10430 Jakarta, Indonesia. Medicines for Malaria Venture, PO Box 1826, 20 route de Pre-Bois,
215 Geneva 15, Switzerland. Swiss Tropical and Public Health Institute, Socinstrasse 57, 4051 Basel, Switzerland. Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine,
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University of Oxford, Oxford OX3 7LJ, UK. Division of Infectious Diseases, Department of Medicine, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.
{Present address: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.
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RESEARCH ARTICLE
Hit from screen
DDD102542
DDD103679
DDD107498
Pf(3D7) EC50 0.12 μM
Calculated log(P) 4.3
Solubility not determined
Pf(3D7) EC50 0.35 μM
Calculated log(P) 3.7
Solubility 36 μM
Pf(3D7) EC50 0.70 μM
Calculated log(P) 3.7
Solubility 180 μM
Pf(3D7) EC50 0.001 μM
Calculated log(P) 3.2
Solubility 216 μM
–1
Mouse cli. 5.3 ml min g–1
–
1
Mouse cli. 8.6 ml min g–1
–1
Mouse cli. 3.4 ml min g–1
–1
Mouse cli. < 0.9 ml min g–1
Figure 1
|
Chemical evolution of DDD107498 from the phenotypic hit. Calculated log(P), calculated log(partition coefficient); Solubility, solubility in water;
Mouse cli., intrinsic clearance in mouse liver microsomes.
P. vivax (median EC50 5 0.51 nM (range 0.25–1.39 nM), n 5 28)
collected from patients with malaria from southern Papua, Indonesia,
a region where high-grade multidrug-resistant malaria is endemic for
a
Treatment
1
0
DDD107498 (mg per kg)
Vehicle
0.1
7,8
both species (Extended Data Fig. 2b) . In contrast, the compound was
not toxic to human cells (MRC5 and Hep-G2 cells) at much higher
concentrations (.20,000-fold selectivity; Extended Data Fig. 2c).
DDD107498 showed good drug-like properties: metabolic stability
when incubated with hepatic microsomes or hepatocytes from several
species; good solubility in a range of different media; and low protein
binding (Tables 1 and 2 in Supplementary Methods). DDD107498
displayed excellent pharmacokinetic properties in preclinical species,
including good oral bioavailability (an important prerequisite for
use in resource-poor settings) and long plasma half-life (important
for single-dose treatment and chemoprotection) (Extended Data
Table 1a).
1
90% reduction
0
0
1
3
.3
.6
0.1
.01
Limit of detection
0
0
1
2
3
4
5
6
7
Time after infection (days)
b
6
5
4
3
2
1
0
DDD107498
Atovaquone
Pyrimethamine
Chloroquine
Artemisinin
DDD107498 was very active in several mouse models of malaria,
with comparable or greater efficacy than current antimalarials
(Extended Data Table 1b). DDD107498 had a 90% reduction in
parasitaemia (ED ) of 0.57 mg per kg (body weight) after a single
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0
oral dose in mice infected with the rodent parasite Plasmodium
berghei. Efficacy was also tested in NOD-scid IL-2R_null mice
engrafted with human erythrocytes and infected with P. falciparum
0
24
48
72
96
120
0
087/N9
9
strain 3D7
(Fig. 2a) . When orally dosed daily for 4 days, the
Treatment time (h)
c
ED90 on day 7 after infection was 0.95 mg per kg per day. Blood
sampling from the infected SCID (severe combined immunodefi-
ciency) mice suggested a minimum parasiticidal concentration for
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3
2
1
0
DDD107498
1
3
1
0
0 × EC50
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DDD107498 of 10–13 ng ml for asexual blood-stage infections.
The effects of DDD107498 on circulating parasites in the SCID
mouse model could be observed in one replication cycle (48 h) and
led to trophozoites with condensed cytoplasm (Extended Data Fig. 3).
Stage specificity studies using synchronized cultures showed that, at a
concentration of 4 nM for 24 h, DDD107498 led to (1) from the ring
stage, formation of abnormal trophozoites; (2) from the trophozoite
stage, prevention of schizont formation with a 50% reduction in para-
sites, indicative of cidal activity; and (3) from the schizont stage,
× EC50
× EC50
.3 × EC50
0.1 × EC50
0
24
48
72
96
120
Treatment time (h)
prevention of ring formation with a 98% reduction in parasites, indi- Figure 2
|
Efficacy studies and parasite killing rate. a, In vivo activity against
P. falciparum in NOD-scid IL-2R_null mice. Six mice were used, each serially
sampled. The variability of cytometry for repeated acquisitions of a sample
is less than 2–3%. The percentage of parasitaemia was calculated by acquiring a
minimum numberof 500parasitizederythrocytes. An independent experiment
with three further mice was performed to confirm the ED90. b, Determination
of the in vitro killing rate of DDD107498. The in vitro parasite reduction rate
cative of cidal activity (Extended Data Fig. 3b, c).
DDD107498 showed a similar parasite killing profile both in vitro
(Fig. 2b, c) and in vivo (Fig. 2a), which is supportive of a common
mode of action in cellular and animal models of disease. Using a
10
parasite reduction rate assay there was a lag of about 24–48 h, during
which time the effects of the compounds were reversible following assay was used to determine onset of action and rate of killing as previously
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0
wash-out. Rapid killing occurred after parasites had been exposed to described . P. falciparum was exposed to DDD107498 at a concentration
DDD107498 for more than 48 h (Fig. 2b, c).
corresponding to 10 3 EC50. The number of viable parasites at each time point
10
was determined as described . Four independent serial dilutions were done
with each sample to correct for experimental variation; error bars, s.d. Previous
results reported on standard antimalarials tested at 10 3 EC50 using the same
All these experiments suggest for the blood-stage form that treat-
ment with DDD107498 prevented development of trophozoites and
schizonts and, at least in the case of schizonts, caused rapid killing.
Any ring-stage parasites only developed as far as abnormal tropho-
zoites, which appeared to survive for about 48 h under drug pressure,
but were then killed.
1
0
conditions are shown for comparison . c, The in vitro parasite reduction
rate assay was used to determine the minimal concentration of compound
needed for achieving maximal killing effects. Parasites were exposed to
DDD107498 at concentrations of 0.1, 0.3, 1, 3 and 10 3 EC50 using conditions
In safety studies, DDD107498 showed no clinically relevant inhibi- described above. Error bars, s.d. Concentrationsof DDD107498of 13 EC50 are
tion of any of the major human cytochrome P450 (CYP) isoforms and sufficient to produce maximal killing effects on treated parasites.
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ARTICLE RESEARCH
0
h.p.i.
2 h.p.i.
24 h.p.i.
No treatment
48 h.p.i.
Compound
*
Compound
Compound
No treatment
No treatment
*
No treatment
No treatment
Compound
*
No treatment
Compound
Atovaquone
DDD107498
Compound
EC50 (nM)
Figure 3
|
In vitro activity of DDD107498 against P. berghei liver stages. Each experiment was the average of four technical replicates; h.p.i., hours
post-infection. Bars, 95% CI. *More than one curve fitting possible.
CYP450 induction risk was low (Table 3 in Supplementary Methods), after 7 days) with an EC50 of 1.8 nM. At a baseline oocyst intensity of
indicating a low risk of clinical drug–drug interactions. DDD107498 27 oocysts per mosquito in the DMSO controls, prevalence of infec-
is non-mutagenic and has very weak inhibitory potencies on IKr tion was inhibited, with an EC50 of 3.7 nM, as measured by the
(hERG) and other ion channels, indicating a very low risk for adverse number of infected mosquitoes. Repeating the standard membrane
cardiovascular activity. Given its potency, long half-life observed in feeding assay in which DDD107498 was added at the moment of
preclinical species, and safety margins from a rat 7-day toxicology mosquito feeding gave an EC50 of 10 nM, indicating potent activity
study, DDD107498 has potential both for single-dose treatment and against the parasite sexual stages that develop in the mosquito midgut
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for once-weekly chemoprotection .
(Extended Data Fig. 5) .
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7,18
A P. berghei mouse-to-mouse
model was additionally used to
Activity against other life-cycle stages
Intra-hepatocytic parasites (liver schizont stages) are the first stage of (PbGFPCON507) , and then treated orally with compound 24 h
examine transmission blockade. Mice were infected with P. berghei
19
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7
human infection after injection of sporozoites by anopheline mosqui- before mosquitoes took a direct blood meal . After a 3 mg per kg
toes. Compounds active against this stage have potential for use dose of DDD107498, a 90.7% reduction in infected mosquitoes and a
in chemoprotection. DDD107498 showed an EC50 < 1 nM against 98.8% reduction in oocysts per midgut was observed at day 10 com-
the liver schizont forms of P. berghei and Plasmodium yoelii . pared with mosquitoes fed on untreated mice (Extended Data Fig. 6).
DDD107498 was active when dosed for only 2 h during the initial A corresponding reduction in sporozoite intensity and prevalence was
infection (hepatocyte invasion) of the liver cells (Fig. 3). In contrast, observed (93.8% and 88.6% respectively). Mosquitoes previously fed
atovaquone (clinically used for chemoprotection in combination on infected, drug-treated mice were allowed to feed on uninfected
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2
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with proguanil) had a much reduced activity during this period mice . Testing a range of mosquito biting rates, we observed a mean
EC50 < 106 nM versus 0.3 nM for continuous treatment). Further, reduction of 89.5% (95% CI 71.4–100) in the number of mice that
(
DDD107498 showed equivalent potency against the P. berghei liver developed blood-stage infection compared with mice bitten by mos-
stage when added after initial infection had been established (Fig. 3). quitoes that had fed on non-drug-treated infected mice. The overall
This suggests that intermittent treatment may be sufficient for che- effectiveness of an intervention over a round of transmission (from
moprotection. To assess chemoprotective potential in vivo, mice were mouse to mosquito to mouse) can be quantified by estimating its
treated with DDD107498 2 h before being infected with luciferase- ability to reduce the basic reproductive number (R ). This has been
0
expressing P. berghei sporozoites (Extended Data Fig. 4). At a dose of 3 termed the ‘effect size’ of an intervention. By fitting data from the
mg per kg, DDD107498 was fully curative with no sign of parasitae- mouse-to-mouse assay to a chain binomial model we can estimate the
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mia after 30 days. Thus, DDD107498 demonstrates potent chemo- effect size of the intervention , assessing the ability of DDD107498
protection using in vitro and in vivo models, where blood sampling usage to reduce the basic reproductive number R (assuming 100%
from the mice during the experiment suggests a minimum parasiti- coverage). Our results estimate an effect size of 90.5% (95% CI
0
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cidal concentration of 15–20 ng ml
.
78.3–94.2), suggesting that DDD107498 is capable of acting as a
The parasite erythrocytic form differentiates into the asymptomatic potent transmission-blocking drug over multiple transmission set-
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male and female gametocytes (stages I–V) within the human host. tings within a field context . The combination of these key in vitro
Mature stage V gametocytes are infective to mosquitoes, but are and in vivo assays demonstrates the very strong potential of
not eliminated by the majority of current antimalarial agents, and DDD107498 for blocking transmission; importantly, the required
remain circulating in the human bloodstream for up to 3 weeks, long doses are likely to be similar to those required for treatment of
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after the disappearance of clinical symptoms of malaria . After blood-stage malaria.
ingestion by the mosquito, gametocytes differentiate into gametes.
DDD107498 targets PfeEF2
DDD107498 potently inhibited both male and female gamete forma-
tion from the gametocyte stage at similar concentrations (1.8 nM To determine the molecular target for DDD107498, asexual blood-
(
95% CI 1.6–2.1 nM) and 1.2 nM (95% CI 0.8–1.6 nM) respectively; stage P. falciparum were cultured in the presence of DDD107498 at
20
Extended Data Fig. 5), indicating that it is an extremely potent inhib- 5 3 EC , until parasites became resistant (Extended Data Table 2) .
50
itor of the functional viability of both male and female mature game- Resistance was obtained in the 3D7 (drug-sensitive) and 7G8 and Dd2
1
5
7
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tocytes . In line with this, DDD107498 blocked transmission, as (multi-drug-resistant) strains, with minimum inocula of 10 , 10 and
6
determined by the standard membrane feeding assay. In this assay, 10 parasites respectively. Genomic DNA was extracted from resistant
parasite cultures containing P. falciparum stage V gametocytes were lines and whole-genome sequencing of ten drug-resistant lines iden-
exposed to compound for 24 h before mosquito feeding. DDD107498 tified shared mutations in one gene, which were not present in the
blocked subsequent oocyst development in the mosquito (measured parental lines: Pf3D7_1451100 (Supplementary Data 1). This gene
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RESEARCH ARTICLE
a
Ribosome
tRNA
b
GTP
GDP
eEF2
I183T (3D7)
T185I (7G8)
5
′
3′
mRNA
Translocation
+Hoechst
E134G (3D7)
E134D (Dd2)
P754A (3D7)
P754S (3D7)
eEF2–GFP
DIC
c
d
L755F (Dd2)
Y186N (Dd2)
A482T (Dd2)
S474R (7G8)
5
μm
Protein synthesis inhibition
DDD107498
Cycloheximide
Actinomycin D
1
1
20
00
120
100
80
120
100
80
8
6
4
2
0
0
0
0
0
60
60
40
40
20
20
0
0
–
20
–20
–20
0.01 0.1
1
10 100 1,000
0.01 0.1
1
10 100 1,000
0.01 0.1
1
10 100 1,000
Inhibitor, fold EC50 value
Inhibitor, fold EC50 value
Inhibitor, fold EC50 value
DNA/RNA synthesis inhibition
DDD107498
Cycloheximide
Actinomycin D
1
20
00
120
100
80
120
1
100
8
0
0
8
6
4
2
0
0
0
0
0
6
60
40
20
0
4
0
0
0
2
–
20
–
20
–20
0.01 0.1
0
.01 0.1
1
10 100 1,000
1
10 100 1,000
0.001 0.01 0.1
1
10
Inhibitor, fold EC50 value
Inhibitor, fold EC50 value
Inhibitor, fold EC50 value
e
f
120
0
.5
1
00
80
60
40
0
0
0
0
.4
.3
.2
.1
0
Dd2
DDD-R
*
**
**
DDD-R + eEF2-WT
DDD-R + eEF2-Y186N
2
0
0
–
20
0.01
0.1
1
10
100 1,000 10,000 1 × 105
DDD107498 (nM)
Figure 4
|
DDD107498 targets protein synthesis via eEF2. a, eEF2 promotes DDD107498, cycloheximide or actinomycin D. Radiolabelled incorporation,
the GTP-dependent translocation of the ribosome along messenger RNA
measured as counts per minute, was normalized as the percentage of
during protein synthesis. b, Homology model of P. falciparum eEF2. The
incorporation against inhibitor concentration (means 6 s.d.; n 5 3 indepen-
mapped mutations from each strain are colour coded by EC50 fold (red, high; dent experiments, each run in duplicate). e, The EC50 values for transfectants
amber, moderate; green, low). c, Live-cell imaging of P. falciparum expressing against DDD107498 (means 6 s.d.; n 5 4–7 independent experiments,
an extra copy of eEF2 (WT) fused to GFP. The image is representative of more each run in duplicate). Statistical significance was determined by the Mann–
than 50 parasites visualized on two independent occasions. d, Protein and
Whitney U-test: *P , 0.05; **P , 0.01. f, DDD107498-resistant line
(eEF2-Y186N) transfected episomally with plasmids expressing either WT
eEF2 or eEF2-Y186N (means 6 s.d.; n 5 3 independent experiments, each
run in duplicate).
DNA/RNA synthesis were evaluated by measuring the incorporation of
35
3
[
S]Met/Cys (upper panel) and [ H]hypoxanthine (lower panel) into
asynchronous 3D7 wild-type (open circles) and 3D7 DDD107498-resistant
lines (eEF2-E134A/P754A) (filled circles) after incubation for 40 min with
encodes P. falciparum translation elongation factor 2 (PfeEF2). Three to DDD107498 can be associated with multiple independent muta-
lines had two single nucleotide polymorphisms in PfeEF2, with a tions is in keeping with observations from both artemisinin and other
2
1–23
mixture of wild-type (WT) and mutant reads at each position antimalarial compounds in development
Supplementary Data 2), suggesting that these lines were mixtures
eEF2 is one of several essential elongation factors required in eukar-
of two clones, each with independent mutations in PfeEF2. Single yotic protein synthesis, by mediating GTP-dependent translocation of
.
(
2
4
nucleotide polymorphisms were confirmed by Sanger sequencing the ribosome along messenger RNA (mRNA) (Fig. 4a) . Yeast eEF2 is
2
5,26
and nine validated mutations in PfeEF2 clustered in three regions the target of the antifungal compound sordarin . Sordarin is select-
of the encoded protein. In two cases identical single nucleotide poly- ive for the Saccharomyces cerevisiae eEF2 over mammalian eEF2 in
morphisms were identified in two independent lines, indicating vitro, demonstrating that although protein synthesis in eukaryotes is
mutations in functionally important residues that were acquired inde- conserved, it is possible to obtain selective inhibitors, despite the
pendently in separate selection experiments. The fact that resistance relatively high homology (67.2% identity) between the yeast and
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ARTICLE RESEARCH
25,27
human eEF2 (Extended Data Fig. 7) . In keeping with the potential a high degree of PfeEF2 sequence conservation in the field. The sole
for selectivity, DDD107498 is not toxic to mammalian cells (Extended non-synonymous single nucleotide polymorphism (T16S) identified
Data Fig. 2c). The PfeEF2 mutations associated with resistance was unique to West Africa (allele frequency of 0.002) and is in a
mapped to several areas on the surface of a homology model of the PfeEF2 domain distinct from mutations associated with in vitro resist-
protein (on the basis of the structure of S. cerevisiae eEF2 with no ance to DDD107498.
2
8
ligands bound ), with the mutations giving the highest degree of
resistance clustering together (Fig. 4b). A DDD107498 binding pocket Conclusion
could not be elucidated through these modelling studies.
DDD107498 represents a promising prospect for development as an
We confirmed that brief pre-incubation with DDD107498 specif- antimalarial agent, with a potent activity profile against multiple life-
ically inhibited P. falciparum protein synthesis, by measuring short- cycle stages (sub-10 nM), a novel mode of action and excellent drug-
3
5
term incorporation of [ S]-labelled methionine and cysteine like properties. It has potential for single-dose treatment, which
3
5
(
[ S]Met/Cys) in WT and DDD107498-resistant 3D7 P. falciparum has major implications for ensuring patient compliance and
21
parasites (Fig. 4d) . Cycloheximide (a protein synthesis inhibitor) practical deployment. Its complementary activity on the sexual
3
5
and DDD107498 both prevented [ S]Met/Cys incorporation into stages of the parasite has potential to reduce transmission and its
WT P. falciparum 3D7, whereas actinomycin D (an inhibitor of action on the liver stage suggests a possible role in chemoprotection.
RNA transcription) had minimal effect. Notably, DDD107498 was Chemoprotection and transmission-blocking properties are fun-
1
00-fold less effective at inhibiting protein synthesis in DDD107498- damental to the goal of eliminating and eradicating malaria, for which
resistant 3D7 than WT parasites, whereas cycloheximide prevented the high potency and long half-life of DDD107498 are well suited.
3
5
[
S]Met/Cys incorporation equally in resistant and WT lines.
Owing to general concerns about the emergence of resistance, all
DDD107498 and cycloheximide had minimal effects on DNA/RNA antimalarials are developed as combination therapies, a strategy
biosynthesis both in sensitive and in drug-resistant parasites shown to improve efficacy and reduce the development of drug res-
3
(
measured by incorporation of [ H]-labelled hypoxanthine), dem- istance. In terms of treatment of the erythrocytic stage, DDD107498
onstrating specificity. In contrast, actinomycin D caused a marked fulfils the criteria as a long-duration partner to complete the clearance
3
11
dose-dependent reduction in [ H] incorporation.
of blood-stage parasites . Therefore, it should be combined with a
To confirm that PfeEF2 is the target for DDD107498, we integrated fast-acting compound, ideally with a pharmacological duration of
transgenes expressing either WT PfeEF2 or resistance-associated action as close to DDD107498 as possible. This would reduce the
alleles of PfeEF2 (Y186N, observed in mutant Dd2; and P754S, initial level of infection, with the prolonged activity of DDD107498
1
1
observed in mutant 3D7) using attPxattB integrase-mediated recom- eliminating the remaining parasites .
2
1,29
bination . These transfectants also express endogenous PfeEF2.
Inhibition of protein synthesis by DDD107498 through PfeEF2,
34
Imaging of green fluorescent protein (GFP)-fusions of PfeEF2 showed which is expressed in multiple life-cycle stages , provides mechanistic
cytoplasmic localization (Fig. 4c), indicating that DDD107498 inhi- support for the observed broad-spectrum profile. This highlights
bits protein synthesis in the cytoplasm as opposed to the apicoplast , PfeEF2 as a novel drug target in malaria, and implies that inhibition
the site of action of tetracycline and azithromycin.
of protein synthesis is an effective intervention for achieving multi-
3
0
Dose–response assays with DDD107498 showed a similar inhibi- stage activity in Plasmodium. DDD107498 has now been progressed
tion profile between PfeEF2 transgene-expressing lines and the WT into advanced non-clinical development, with the aim of entering into
Dd2 strain, indicating that the endogenous WT PfeEF2 has a dom- human clinical trials.
inant effect in these experiments (Fig. 4e). This may be due to stable
complex formation between the ribosome, WT PfeEF2 and
Received 10 October 2014; accepted 7 April 2015.
DDD107498, resulting in ribosome stalling, which would explain
why in mixed populations the WT PfeEF2 is dominant. For example
in bacteria, fusidic acid binds to the complex between EF-G and the
ribosome, preventing dissociation and blocking protein translation .
To determine whether WT PfeEF2 was dominant in poisoning
translation, we introduced episomal plasmids encoding WT or
Y186N mutant PfeEF2 into the resistant PfeEF2 Y186N line
1.
2.
3.
4.
5.
World Health Organization. World Malaria Report 2014 (World HealthOrganization,
2014).
Ariey, F. et al. A molecular marker of artemisinin-resistant Plasmodium falciparum
malaria. Nature 505, 50–55 (2014).
Alonso, P. L. et al. A research agenda for malaria eradication: drugs. PLoS Med. 8,
e1000402 (2011).
Wells, T. N. C. & Gutteridge, W. E. in Neglected Diseases and Drug Discovery (eds
Palmer, M. J. & Wells, T. N. C.) Ch. 1 1–32 (Royal Society of Chemistry, 2012).
Brenk, R. et al. Lessons learnt from assembling screening libraries for drug
discovery for neglected diseases. ChemMedChem 3, 435–444 (2008).
3
1
(
Fig. 4f). Plasmid-borne PfeEF2-Y186N had no effect on sensitivity
to DDD107498 (EC50 < 3,100 nM), whereas WT PfeEF2 restored 6. Delves, M. etal.The activities ofcurrentantimalarialdrugs onthe life cycle stages of
Plasmodium: a comparative study with human and rodent parasites. PLoS Med. 9,
e1001169 (2012).
sensitivity (EC50 5 2 nM). This demonstrated a dominant effect of
WT PfeEF2 on parasite susceptibility and confirmed that PfeEF2 is
the primary molecular target of the compound. We note that the
shallow slope observed for the WT PfeEF2 transfected line is probably
a result of heterogeneous episomal plasmid copy number seen with
episomally transformed parasite lines . Structural studies will be
required to define precisely how DDD107498 interacts with eEF2
and the ribosome.
7
8
9
.
.
.
Russell, B. et al. Determinants of in vitro drug susceptibility testing of Plasmodium
vivax. Antimicrob. Agents Chemother. 52, 1040–1045 (2008).
Karyana, M. et al. Malaria morbidity in Papua Indonesia, an area with multidrug
resistant Plasmodium vivax and Plasmodium falciparum. Malar. J. 7, 148 (2008).
Angulo-Barturen, I. et al. A murine model of falciparum-malaria by in vivo selection
of competent strains in non-myelodepleted mice engrafted with human
erythrocytes. PLoS ONE 3, e2252 (2008).
29
1
0. Sanz, L. M. et al. P. falciparum in vitro killing rates allow to discriminate between
different antimalarial mode-of-action. PLoS ONE 7, e30949 (2012).
Resistance has been reported for all clinical antimalarials, including 11. Burrows, J. N., van Huijsduijnen, R. H., Mohrle, J. J., Oeuvray, C. & Wells, T. N.
2
Designing the next generation of medicines for malaria control and eradication.
Malar. J. 12, 187 (2013).
artemisinins . While the correlation between the rate of resistance
generation in laboratory and clinical settings for antimalarials is not
fully understood, it is important to evaluate the risk of all new anti-
1
2. Meister, S. et al. Imaging of Plasmodium liver stages to drive next-generation
antimalarial drug discovery. Science 334, 1372–1377 (2011).
malarials both in preclinical and in clinical studies. In our studies, the 13. Adjalley, S. H. et al. Quantitative assessment of Plasmodium falciparum sexual
development reveals potent transmission-blocking activity by methylene blue.
minimum inoculum for generating resistance to DDD107498 is
Proc. Natl Acad. Sci. USA 108, E1214–E1223 (2011).
32
within acceptable limits . Furthermore, selected resistant Dd2 lines
revealed impaired growth rates in the absence of drug pressure com-
pared with WT Dd2 (Extended Data Fig. 8); moreover the higher the
resistance, the greater the fitness defect. Importantlygenomesequences
1
4. Bousema, T. & Drakeley, C. Epidemiology and infectivity of Plasmodium falciparum
and Plasmodium vivax gametocytes in relation to malaria control and elimination.
Clin. Microbiol. Rev. 24, 377–410 (2011).
1
5. Delves, M. J. et al. Male and female Plasmodium falciparum mature gametocytes
show different responses to antimalarial drugs. Antimicrob. Agents Chemother. 57,
3268–3274 (2013).
2
3,33
from 1,685 clinical isolates ofP. falciparum from 17 countries reveal
1
8 J U N E 2 0 1 5 | V O L 5 2 2 | N A T U R E | 3 1 9
G2015 Macmillan Publishers Limited. All rights reserved

RESEARCH ARTICLE
1
1
1
6. Delves, M. J. et al. A high-throughput assay for the identification of malarial
transmission-blocking drugs and vaccines. Int. J. Parasitol. 42, 999–1006 (2012).
7. Blagborough, A. M. et al. Transmission-blocking interventions eliminate malaria
from laboratory populations. Nature Commun. 4, 1812 (2013).
8. Upton, L. M. et al. Lead clinical and preclinical antimalarial drugs can significantly
reduce sporozoite transmission to vertebrate populations. Antimicrob. Agents
Chemother. 59, 490–497 (2015).
32. Ding, X. C., Ubben, D. & Wells, T. N. A framework for assessing the risk of resistance
for anti-malarials in development. Malar. J. 11, 292 (2012).
33. Manske, M. et al. Analysis of Plasmodium falciparum diversity in natural infections
by deep sequencing. Nature 487, 375–379 (2012).
34. Florens, L. et al. A proteomic view of the Plasmodium falciparum life cycle. Nature
419, 520–526 (2002).
3
5. Jim e´ nez-D ´ı az, M. B. et al. Quantitative measurement of Plasmodium-infected
erythrocytes in murine models of malaria by flow cytometry using bidimensional
assessment of SYTO-16 fluorescence. Cytometry A 75, 225–235 (2009).
6. Charman, S. A. etal.Synthetic ozonide drugcandidateOZ439 offersnew hopefor a
single-dose cure of uncomplicated malaria. Proc. Natl Acad. Sci. USA 108,
1
9. Janse, C. J. etal. High efficiency transfection ofPlasmodiumberghei facilitates novel
selection procedures. Mol. Biochem. Parasitol. 145, 60–70 (2006).
20. Flannery, E. L., Fidock, D. A. & Winzeler, E. A. Using genetic methods to define the
targets of compounds with antimalarial activity. J. Med. Chem. 56, 7761–7771
3
(2013).
4
400–4405 (2011).
2
2
2
2
2
2
1. Rottman, M. et al. Spiroindolones, a potent compound class for the treatment of
malaria. Science 329, 1175–1180 (2010).
Supplementary Information is available in the online version of the paper.
2. McNamara, C. W. et al. Targeting Plasmodium PI(4)K to eliminate malaria. Nature
504, 248–253 (2013).
Acknowledgements This work was supported by grants from Medicines for Malaria
Venture, the Wellcome Trust (100476 (I.H.G., A.H.F.), 091625 (R.N.P.) and 098051
3. Miotto, O. et al. Multiple populations of artemisinin-resistant Plasmodium
falciparum in Cambodia. Nature Genet. 45, 648–655 (2013).
4. Jorgensen, R., Merrill, A. R. & Andersen, G. R. The life and death of translation
elongation factor 2. Biochem. Soc. Trans. 34, 1–6 (2006).
5. Justice, M. C. et al. Elongation factor 2 as a novel target for selective inhibition of
fungal protein synthesis. J. Biol. Chem. 273, 3148–3151 (1998).
6. Capa, L., Mendoza, A., Lavandera, J. L., de las Heras, F. G. & Garcia-Bustos, J. F.
Translation elongation factor 2 is part of the target for a new family of antifungals.
Antimicrob. Agents Chemother. 42, 2694–2699 (1998).
(
J.C.R., W.P., T.D.O.)), the Bill andMelinda Gates Foundation (OPP1043501 (M.D., R.S.)),
the National Institutes of Health (R01 AI103058 to E.A.W. and D.A.F.) and the European
Union (EVIMalaR (T.D.O.)). Drug Discovery Unit infrastructure was supported by the
European Regional Development Fund 2007-2013 and UK Research Partnership
Investment Fund awards to M. Ferguson, whom we also thank for continued support.
We thank C. Sibley for discussions. We acknowledge the East Scotland Blood
Transfusion Service, Ninewells Hospital, Dundee, for erythrocyte supply to Dundee. We
thank L. D. Shultz and The Jackson Laboratory for providing access to non-obese
diabetic SCID IL2Rcc null mice through their collaboration with the GlaxoSmithKline
Tres Cantos Medicines Development Campus. The following are acknowledged for
technical assistance: all members of the Drug Discovery Unit (Dundee), M. Berriman,
J. Kamber, E. Kenangalem, A. LaCrue, O. Montagnat, J. Rini Poespoprodjo, M. Sanders,
S. Sax, C. Scheurer, L. Trianty, M. Tunnicliff (detailed in Supplementary Information).
2
2
2
3
3
7. Shastry, M. etal.Species-specific inhibitionoffungalproteinsynthesisbysordarin:
identification of a sordarin-specificity region in eukaryotic elongation factor 2.
Microbiology 147, 383–390 (2001).
8. Jorgensen, R. et al. Two crystal structures demonstrate large conformational
changes in the eukaryotic ribosomal translocase. Nature Struct. Biol. 10, 379–385
(2003).
9. Nkrumah, L. J. et al. Efficient site-specific integration in Plasmodium falciparum
chromosomes mediated by mycobacteriophage Bxb1 integrase. Nature Methods
Author Contributions The author contributions are detailed in the Supplementary
Information.
3, 615–621 (2006).
0. Biswas, S. et al. Interaction of apicoplast-encoded elongation factor (EF) EF-Tu with
nuclear-encoded EF-Ts mediates translation in the Plasmodium falciparum plastid.
Int. J. Parasitol. 41, 417–427 (2011).
1. Cox, G. et al. Ribosome clearance by FusB-type proteins mediates resistance to the
antibiotic fusidic acid. Proc. Natl Acad. Sci. USA 109, 2102–2107 (2012).
Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare competing financial interests: details
are available in the online version of the paper. Readers are welcome to comment on
the online version of the paper. Correspondence and requests for materials should be
addressed to I.H.G. (i.h.gilbert@dundee.ac.uk) or K.D.R. (k.read@dundee.ac.uk).
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Extended Data Figure 1
|
Synthetic methodology. a, Synthesis of DDD107498 (4). b, Synthesis of DDD102542 (6a) and DDD103679 (6b).
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RESEARCH ARTICLE
3
Extended Data Figure 2
|
In vitro activity. a, In vitro activity against a panel independent [ H]hypoxanthine incorporation experiments (each run in
of resistant and sensitive strains of P. falciparum. CQ, chloroquine; PYR,
pyrimethamine; CYC, cycloguanil; QUI, quinine; SUL, sulfadoxine; MFQ,
mefloquine. Resistance as follows: K1 (CQ, SUL, PYR, CYC); W2 (CQ, SUL,
PYR, CYC); 7G8 (CQ, PYR, CYC); TM90C2A (CQ, PYR, MFQ, CYC); D6
duplicate). b, Ex vivo activity against P. falciparum and P. vivax clinical isolates
from Papua (Indonesia). The lines on the scatter plots represent the median
value for each drug. c, Effect of DDD107498 on P. falciparum 3D7, HepG2 and
MRC5 cells. Data are the means 6 s.d. of n reported independent experiments.
(MFQ); V1/S (CQ, SUL, PYR, CYC). Data are the means 6 s.d. of n 5 3
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ARTICLE RESEARCH
Extended Data Figure 3
|
Effect of DDD107498 on parasite morphology.
at this stage. The images displayed are taken from a mouse with high levels of
a, Phenotypeof P. falciparum in peripheral blood of NOD-scid IL-2R_null mice parasitaemia. At least 50 parasites were counted per sample screened in the
engrafted with human erythrocytes. Blood samples were taken at days 5 and 7 microscope. Of these, four photographs of representative parasite phenotypes
oftheassay(one andtwoasexual cycles, respectively)after thestartoftreatment were selected to represent the morphology of the most prevalent phenotype.
with vehicle or DDD107498 at day 3. The bi-dimensional flow cytometry plots Thus, this is a qualitative assessment. b, Stage specificity assays using
measure the murine (Ter-119-PE1) and human (Ter-119-PE2) erythrocytes, synchronized cultures. For morphological analysis of antimalarial drug action,
and the presence of nucleic acids (infected SYTO-161 events). The blue circles thin blood smears were prepared, fixed and stained with Giemsa followed by
indicate the region of infected erythrocytes. Vehicle-treated mice showed a examination with an upright microscope using an oil-immersion lens (3100).
characteristic pattern of staining with SYTO-16 (ref. 35), which correlated with For parasitaemia determination, a total number of 1,000 red blood cells
the presence of healthy rings, trophozoites and schizonts in blood smears.
Conversely, mice treated with DDD107498 at 50 mg per kg showed only
(corresponding to five microscopic fields) were counted. R to T, abnormal
trophozoites observed after exposure for 24 h of synchronized rings to
trophozoites with condensed cytoplasm and some pyknotic cells at day 5 (red DDD107498. T to S, trophozoites do not develop into schizonts after exposure
circle in flow cytometry plot and corresponding blood smears). By day 7, few for 24 h to DDD107498. S to R, no ring stages are observed 24 h after treatment
infected erythrocytes were detected by flow cytometry and blood smears
revealed parasites with a similar morphology to those at day 5. This suggests
that trophozoitesarethemost sensitivepopulation since thecycleis interrupted
of schizonts with DDD107498. c, Percentageparasitaemia in theredblood cells.
R, ring stage; T, trophozoite; S, schizont.
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Extended Data Figure 4
|
Prophylactic activity of DDD107498 against sporozoite challenge. P. berghei (luciferase) sporozoite in vivo mouse model of
chemoprotection. A dose of 3 mg per kg was fully protective. Data are the mean of n 5 5 experiments.
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ARTICLE RESEARCH
1
6
Extended Data Figure 5
|
DDD107498 in vitro activity on the different
vitro against P. berghei ookinetes (5.0 nM (95% CI 4.4–5.7 nM)), confirming
life-cycle stages of Plasmodium spp. For comparator data with known
that if DDD107498 was taken up during a blood meal, it could continue to kill
antimalarials, see figure 5 in ref. 6. DDD107498 also showed potent activity in parasites within the mosquito.
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RESEARCH ARTICLE
Extended Data Figure 6
a, Impact of DDD107498 treatment on in vivo mosquito infection. Mice
infected with P. berghei (PbGFPCON507 clone 1) were dosed orally with
DDD107489 at 3 mg per kg, atovaquone at 0.3 mg per kg, sulphadiazine at
|
In vivo P. berghei mouse-to-mouse assay.
feeds, surviving potentially infectious mosquitoes were allowed to blood-feed
on naive mice at a range of transmission settings (biting rates of 2, 5 and 10 bites
per naive mouse) to assess the ability of drug treatment to reduce the number
of new malarial cases after mosquito bite. Efficacy is expressed as the following:
19
8
.4 mg per kg, or were not drug treated (negative control). After 24 h, popula- (1) impact on the mosquito population—expressed as reduction in both
tions of treated mice (n 5 5) were exposed to 500 female Anopheles stephensi oocyst and sporozoite intensity and prevalence; (2) impact on subsequent
mosquitoes, and oocyst intensity and infection prevalence in the mosquito transmission to newnaivevertebratehosts—expressedas reductionin infection
midgutwas measured10days after feeding. Individualdata pointsrepresentthe of naive mice (reduction in number of new malarial cases after mosquito bite)
number of oocysts found in individual mosquitoes. Two replicates were
performed. Sulphadiazine (8.4 mg per kg, intraperitoneally) and atovaquone
and inhibition of subsequent parasitaemia (day 10 after bite) in mice that do
become infected; (3) effect size generated—by fitting the data achieved within
this assay to a chain-binomial model we could assess the ability of DDD107498
(
0.3 mg per kg, intraperitoneally) were used as negative and positive
transmission-blocking drug controls, respectively. Horizontal bars represent
mean intensity of infection, and error bars s.e.m., within individual mosquito is unsustainable and elimination will occur. Values of 95% CIs are shown in
populations. b, Impact of treatment with DDD107498 over a complete brackets. Efficacy is calculated in comparison with no-drug controls. TB,
transmission cycle in vivo. After drug treatment of infected mice and mosquito transmission blocking.
0 0
to reduce R (assuming 100% coverage). If R is reduced to ,1, transmission
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ARTICLE RESEARCH
Extended Data Figure 7
|
ClustalWS alignment of eEF2 sequences from human, yeast and Plasmodium (P. falciparum). Alignment made using Jalview
ClustalX default colouring.
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Extended Data Figure 8
parasite lines. Unmarked Dd2- and DDD107498-selected parasites with
various levels of resistance were assessed for growth in a competition assay,
|
Fitness phenotypes of DDD107498-resistant
b, Growth assay of four different test lines: (1) WT Dd2, (2) EF2-E134D, (3)
EF2-L755F and (4) EF2-Y186N, relative to Dd2–GFP. A faster growth rate of
WT Dd2 (DDD107498 IC50 0.14 nM) relative to the fluorescent Dd2–GFP line
relative to a Dd2–GFP reference line. a, Equal numbers of unmarked test lines is reflected in an increased ratio over time. The low-level resistant line EF2-
were mixed with the Dd2–GFP reference, in triplicate wells, and the ratio of E134D (IC50 5.8 nM) did not attain a WT growth rate, and the high-level
non-fluorescent and fluorescent cells assessed by flow cytometry over time. At resistant lines EF2-L755F (IC50 660 nM) and EF2-Y186N (3,100 nM) were
day 0, all lines had a 1:1 ratio with the Dd2–GFP reference. Increased growth of further impaired. Means 6 s.d.; n 5 4 independent experiments, each run in
the test line over the Dd2–GFP reference, which has a slower growth rate than triplicate.
unmarked WT Dd2, would result in an increased ratio of test:Dd2–GFP.
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Extended Data Table 1
|
Pharmacokinetics and rodent efficacy of DDD107498
a, In vivo pharmacokinetic parameters in preclinical species (n 5 3, all species). b, Oral in vivo single-dose efficacy of antimalarial drugs and DDD107498 in the murine P. berghei model. Data are the means of n 5 3
for DDD107498 and n $ 5 for the reference compounds. 1Data from ref. 36.
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Extended Data Table 2
|
Summary of resistance selection experiments with DDD107498
*
Number of positive cultures (n 5 3) and day of recrudescence for each inoculum. {EC50 fold change was determined on bulk cultures. MIR, minimum inoculum of resistance.
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