ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 2010, p.

26032610
0066-4804/10/$12.00 doi:10.1128/AAC.01526-09
Copyright 2010, American Society for Microbiology. All Rights Reserved.

Vol. 54, No. 6

Preclinical Evaluation of the Antifolate QN254, 5-ChloroN6-(2,5-Dimethoxy-Benzyl)-Quinazoline-2,4,6-Triamine,

as an Antimalarial Drug Candidate
Alexis Nzila,1,2* Matthias Rottmann,3 Penchit Chitnumsub,4 Stevens M. Kiara,1
Sumalee Kamchonwongpaisan,4 Cherdsak Maneeruttanarungroj,4
Supannee Taweechai,4 Bryan K. S. Yeung,5 Anne Goh,5
Suresh B. Lakshminarayana,5 Bin Zou,5 Josephine Wong,5
Ngai Ling Ma,5 Margaret Weaver,6 Thomas H. Keller,5
Veronique Dartois,5 Sergio Wittlin,3 Reto Brun,3
Yongyuth Yuthavong,4 and Thierry T. Diagana5*
Kenya Medical Research Institute/Wellcome Trust Collaborative Research Program, P.O. Box 230, 80108, Kilifi, Kenya1; University of
Oxford, Nuffield Department of Medicine, John Radcliffe Hospital, Oxford, United Kingdom2; Swiss Tropical Institute,
Parasite Chemotherapy, Socinstrasse 57, P.O. Box, CH-4002 Basel, Switzerland3; National Centre for
Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, NSTDA, Pathumthani 12120,
Thailand4; Novartis Institute for Tropical Diseases, 10 Biopolis Road #05-01 Chromos, 138670 Singapore5;
and Novartis Institute for Biomedical Research, 250 Massachusetts Ave., Cambridge, Massachusetts 021396
Received 28 October 2009/Returned for modification 11 January 2010/Accepted 19 February 2010

Drug resistance against dihydrofolate reductase (DHFR) inhibitorssuch as pyrimethamine (PM)has

now spread to almost all regions where malaria is endemic, rendering antifolate-based malaria treatments
highly ineffective. We have previously shown that the di-amino quinazoline QN254 [5-chloro-N6-(2,5-dimethoxy-benzyl)-quinazoline-2,4,6-triamine] is active against the highly PM-resistant Plasmodium falciparum V1S
strain, suggesting that QN254 could be used to treat malaria in regions with a high prevalence of antifolate
resistance. Here, we further demonstrate that QN254 is highly active against Plasmodium falciparum clinical
isolates, displaying various levels of antifolate drug resistance, and we provide biochemical and structural
evidence that QN254 binds and inhibits the function of both the wild-type and the quadruple-mutant (V1S)
forms of the DHFR enzyme. In addition, we have assessed QN254 oral bioavailability, efficacy, and safety in
vivo. The compound displays favorable pharmacokinetic properties after oral administration in rodents. The
drug was remarkably efficacious against Plasmodium berghei and could fully cure infected mice with three daily
oral doses of 30 mg/kg. In the course of these efficacy studies, we have uncovered some dose limiting toxicity
at higher doses that was confirmed in rats. Thus, despite its relative in vitro selectivity toward the Plasmodium
DHFR enzyme, QN254 does not show the adequate therapeutic index to justify its further development as a
single agent.

of artemisinin derivatives is reduced in Southeast Asia, where

artemisinin derivatives have been used for a long time as
monotherapies (7, 28, 53). This is a cause of concern since the
spread of artemisinin resistance will compromise the usefulness of ACTs globally. Thus, there is an urgent need to discover and develop new alternative drugs.
For several decades, dihydrofolate reductase (DHFR) has
been targeted with different classes of chemical entities for the
development of new therapies for a broad range of therapeutic
indications, including several parasitic diseases (13). DHFR
catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF), which is an essential cofactor in the biosynthesis
of deoxythymidylate monophosphate (dTMP), a metabolite
essential to DNA synthesis and cell replication.
Pyrimethamine (PM) is a potent inhibitor of the Plasmodium DHFR enzyme, and this compound has been widely used
in combination with the dihydropteroate synthetase (DHPS)
inhibitor sulfadoxine. Unfortunately, PM resistance is now
common, rendering this drug ineffective (30). One of the documented mechanisms of antifolate resistance is through the

Malaria control is a global public health priority that has

been hampered by the rapid development and spread of resistance against antimalarials. As a consequence, the World
Health Organization (WHO) recommends the use of artemisinin-containing combination therapies (ACTs) as a first-line
treatment for malaria. Although ACTs are designed to reduce
the chance of artemisinin drug resistance development, there
are considerable concerns that this may already have occurred.
For instance, there is now mounting evidence that the efficacy

* Corresponding author. Mailing address for A. Nzila: Kenya Medical Research Institute/Wellcome Trust Collaborative Research Program, P.O. Box 230, 80108 Kilifi, Kenya. Phone: (254) 417-522-535.
Fax: (254) 417-522-290. E-mail: anzila@kilifi.kemri-wellcome.org.
Mailing address for T. T. Diagana: Novartis Institute for Tropical
Diseases, 10 Biopolis Road, #05-01 Chromos, Singapore 138670.
Phone: 65 67222988. Fax: 65 63047152. E-mail: thierry.diagana
@novartis.com.
Supplemental material for this article may be found at http://aac
.asm.org/.

Published ahead of print on 29 March 2010.

2603

2604

NZILA ET AL.

mutation of the target itselfthe Plasmodium falciparum

dhfr-ts (Pfdhfr-ts) gene, encoding for a bifunctional enzyme
which possesses both DHFR and thymidylate synthase (TS)
activities carried by two distinct subdomains (29). PM resistance is associated with the mutation of the amino acid Ser to
Asn at codon 108 of DHFR (S108N). Ancillary mutations of
N51I and C59R are associated with an increase in resistance,
and the presence of the mutation I164L results in an even
higher level of PM resistance (14, 47). The presence of these
mutations significantly decreases the sensitivity of the
PfDHFR enzyme to PM inhibition in a biochemical assay (48).
The antifolate triazine WR99210 (3, 22) is potent against P.
falciparum bearing quadruple mutations of DHFR at S108N,
N51I, C59R, and I164L (QM PfDHFR) (20). However,
WR99210 has shown limited efficacy in vivo due to poor oral
bioavailability and displayed some gastrointestinal toxicity. Attempts have been made to circumvent these issues and a prodrug form of WR99210 known as PS-15 has been shown to be
orally active (2). The resolution of the three-dimensional structure of wild-type (WT) and QM PfDHFRwith either PM or
WR99210 bound to its active siteprovided structural insights
into DHFR PM resistance mechanisms, as well as some understanding of the structural features of WR99210 that allow
this compound to retain affinity for QM PfDHFR (55, 56).
The quinazoline pharmacophore has been successfully used
to design drugs for the treatment of cancer and other human
diseases. For example, the DHFR inhibitor, trimetrexate (5-methyl6-[(3,4,5-trimethoxy-phenylamino)-methyl]-quinazoline-2,4diamine) has been developed to treat various cancers in human
patients (15, 32, 33). In a previous study, a series of quinazoline
derivatives was tested against the highly PM-resistant P. falciparum
strain (V1S) and the DHFR inhibitor 2,4-diamino-5-chloro-6-[N(2,5-dimethoxybenzyl)-amino]quinazoline (or QN254 here and compound 1 in reference 34) was found to have potent activitywith an
IC50 (i.e., the inhibitory concentration that reduces parasite growth
by 50% in vitro) of 9 nM (34). Considering the increasingly widespread PM drug resistance, we set out to perform the experiments described here with the goal of further assessing the
potential of QN254 as a candidate antimalarial to replace the
failing PM. Collectively, our data demonstrate that QN254 (i)
binds and inhibits the QM PfDHFR enzyme, (ii) is active on
drug-resistant clinical isolates, and (iii) displays pharmacological properties compatible with an oral antimalarial drug candidate. However, preliminary toxicological findings indicate
that QN254 does not show a therapeutic window sufficiently
large to warrant its progression to the next development stage.
MATERIALS AND METHODS
Drugs and reagents. PM and chloroquine used as standard drugs for evaluation on clinical isolates were purchased from Sigma (Cole, United Kingdom).
WR99210 was a gift from Steve Ward (Liverpool Tropical School, Liverpool,
United Kingdom). In the Ki determination, cycloguanil and WR99210 were gifts
from Tirayut Vilaivan (Chulalongkorn University, Bangkok, Thailand). Standard
drugs for in vivo efficacy testing were obtained from Mepha, Ltd., Switzerland
(artesunate); Sigma (United States) (chloroquine diphosphate); and F. Hoffmann-LaRoche, Ltd., Switzerland (mefloquine hydrochloride). QN254 was prepared as described elsewhere (39).
DHFR biochemical assay and X-ray structure determination. (i) Ki determination. Recombinant PfDHFR enzymes of P. falciparum and human were prepared from Escherichia coli BL21(DE3) bearing pET17b expression plasmids of
P. falciparum WT and quadruple mutant (S108N, N51I, C59R, and I164L) [QM
Pfdhfr] (17), and human dhfr (hdhfr) (18), using a methotrexate column as

ANTIMICROB. AGENTS CHEMOTHER.

described previously (51). Binding affinities (Ki values) of QN254 and standard
antifolate antimalarials, cycloguanil, PM, and WR99210 were determined as
described elsewhere (51).
(ii) X-ray structure determination. Preparation of recombinant PfDHFR-TS
bifunctional enzymes was carried out as previously reported (4). Crystals were
flash-frozen in liquid nitrogen by dipping for 10 s in a corresponding crystallization solution containing 20% glycerol as a cryoprotectant. X-ray diffraction data
were collected under a cold nitrogen stream (100 K) at a wavelength of 1.54
on an FR591 rotating anode X-ray generator equipped with a nonius KappaCCD
detector. The data were processed by using Denzo and Scalepack in the
HKL2000 suite (35). Refinement was performed using a 2.1- 1J3K crystal
structure as a template in both CNS (1) and REFMAC5 (26), along with model
building in program O (16) and model validation in Procheck (24, 25) and
Moleman2 (21). Figures were prepared with PyMOL (6).
Plasmodium strains and culturing methods. (i) Parasite strains and isolates.
We analyzed the in vitro activity of QN254 against clinical isolates from Kenya
with different dhfr genotypes. These clinical isolates were collected in Kilifi
between 2006 and 2008 as part of the malaria studies and were in vitro adapted
for long-term culture as described elsewhere (40).
(ii) In vitro culture and chemosensitivity test. P. falciparum cultures were
carried out in RPMI 1640 (Gibco-BRL, United Kingdom) medium supplemented with 10% (vol/vol) normal human serum, 25 mM bicarbonate, 2 mM
glutamine, 25 mM HEPES buffer, physiological concentrations of para-aminobenzoic acid (5 nM), and folic acid (23 nM). Antimalarial activity was measured
in the presence of various concentrations of each compound by using radioisotopic incorporation (49). The results were expressed as the drug concentration
required for 50% inhibition (IC50) of [3H]hypoxanthine incorporation into parasite nucleic acid, using nonlinear regression analysis of the dose-response curve.
(iii) Parasite genotyping of dhfr. After in vitro adaptation of parasites, infected
blood samples were spotted onto filter paper and stored. Parasite genomic
material from these filter papers was prepared using the methanol procedure and
point mutations at codons 108, 51, 59, and 164 of dhfr were analyzed by PCR and
restriction fragment length polymorphism (PCR-RFLP) enzyme digestion as
described elsewhere (31). Statistical analyses were carried out using Stata, version 9 (StataCorp, College Station, TX), using the Kruskal-Wallis nonparametric
test for comparison of medians. The level of significance was set at P 0.05.
In vivo pharmacokinetic (PK) studies. The Institutional Animal Care and Use
Committee of Novartis Institute for Tropical Diseases, registered with the AgriFood and Veterinary Authority, Government of Singapore, approved all animal
experimental protocols. QN254 was formulated at a concentration of 2.5 mg/ml
for a dose of 25 mg/kg given orally (p.o.) and at 1-mg/ml concentration for a dose
of 5 mg/kg given intravenously (i.v.). The solution formulation for i.v. dosing
contained 10% NMP (n-methyl pyrrolidone), 30% PEG 400, and 60% of 10%
vitamin E-TPGS. The suspension formulation for p.o. dosing was 0.5% carboxymethyl cellulose. For PK studies, blood samples were collected at several time
points after p.o. and i.v. dosing in mice and rats.
(iv) Extraction and LC-MS/MS analysis of QN254. Plasma samples were
extracted with acidified acetonitrile for QN254 using an extractant/plasma ratio
of 8:1. Analyte quantification was performed by liquid chromatography-tandem
mass spectrometry (LC-MS/MS). LC was performed by using an Agilent 1100
HPLC system (Santa Clara, CA), with an Agilent Zorbax XDB Phenyl (3.5 m,
4.6 by 75 mm) column at an oven temperature of 35C, coupled with an API3200
triple quadruple mass spectrometer (Applied Biosystems, Foster City, CA).
Instrument control and data acquisition were performed using Analyst 1.4.2
software (Applied Biosystems). The mobile phases used were phase A (wateracetic acid [99.8:0.2, vol/vol]) and phase B (acetonitrile-acetic acid [99.8:0.2,
vol/vol]), using a gradient, with flow rate of 1.0 ml/min and a run time of 5 min.
Under these conditions, the analyte retention time was 2.9 min. Compound
detection on the mass spectrometer was performed in electrospray positive
ionization mode and using multiple reaction monitoring for specificity, together
with their optimized MS parameters. Analysis of study samples was performed
on different days using an identical method with a ten-point calibration curve
spanning 3 orders of magnitude in compound concentration. The lower and
upper limits of quantification were 12 and 7,500 ng/ml, respectively. Intraday
variability was established with triplicate quality control samples at three concentration levels. The relative standard deviation was under 15%. Interday variability is not reported as calibration curves were reprepared and reanalyzed with
every set of study samples and results accepted if interday variability was found
to be under 15%.
(v) PK data analysis. The mean value from three animals at each time point
was plotted against time to give the plasma concentration-time profile. PK
parameters were determined by using WinNonlin Professional, version 5.0.1
(Pharsight, California), by noncompartmental modeling using software model

VOL. 54, 2010

EVALUATION OF QN254 AS AN ANTIMALARIAL DRUG CANDIDATE

2605

TABLE 1. Results of competitive binding kinetics experiments on DHFR enzymes of P. falciparum WT (WT-PfDHFR) and quadruple
mutant (QM-PfDHFR S108N, N51I, C59R, and I164L) and human (hDHFR) against QN254, WR99210, PM, and cycloguanila
Mean Ki (nM) SD
Agent

CLG
PM
WR99210
QN254
a

Ki ratio

WTPfDHFR

QMPfDHFR

hDHFR

hDHFR/WTpfDHFR

QM-PfDHFR/
WT-PfDHFR

1.51 0.1
0.59 0.05
0.5 0.1
0.39 0.05

454 38
385 163
1.9 0.8
0.58 0.06

55.6 7.8
30.8 1.4
7.7 0.4
10.2 0.6

37
52
15
26

300
652
1.8
1.5

PM, pyrimethamine; CLG, cycloguanil. Ki, binding constant.

200 for oral dosing and model 201 for i.v. dosing. The oral bioavailability (F) was
calculated as the ratio between the area under the curve (AUC) after p.o.
administration and the AUC after i.v. administration corrected for dose (F
AUC p.o. dose i.v./AUC i.v. dose p.o.).
In vivo antimalarial efficacy studies. All in vivo efficacy studies were carried
out at the Swiss Tropical Institute, adhering to the regulations of the veterinary
authorities of Basel-Stadt. The murine P. berghei ANKA was used as previously
described (37). Experimental and control groups of five female NMRI mice (20
to 22 g) were infected i.v. with 108 parasitized erythrocytes/ml, in a volume of 0.2
ml (from a fresh mouse donor). In untreated control mice, parasitemia rises
regularly to 30% by day 3 postinfection and causes death of the animals
between day 5 and day 7 postinfection.
Experimental compounds were prepared in 0.5% carboxymethyl cellulose and
administered orally. Comparators (artesunate, chloroquine, and mefloquine)
were prepared in ETPGS vehicle (10% ethanol, 30% PEG 400, and 60% of a
10% vitamin ETPGS solution) and administered orally. Parasitemia was determined microscopically. The activity was calculated as the difference between
parasitemia for the control and treated groups expressed as a percentage relative
to the control group. The survival time was also recorded up to 30 days after
infection. A compound was considered curative if the animal survived to day 30
after infection with no detectable parasites. The 50% effective dose (ED50) and
ED90 values were assessed at day 3 by plotting the dose as a function of parasitemia, using nonlinear fitting with the Microcal Origin Statistical Program
(OriginLab, Northampton, MA). All data reported are based on 10 mice,
except for the three-times treatment with QN254 which, because of the observed
toxicity, were performed only once with a cohort of five mice.
Rat toxicological study. QN254 was suspended in 10% of a 1% (vol/vol)
aqueous Solutol HS15 solution and 90% of a 0.5% aqueous methylcellulose
M0555 solution and administered to groups of five male rats at daily oral (by
gavage) doses of 50, 150, or 500 mg/kg/day. Animals (Wistar rats; Harlan Laboratories, Ltd., Fu
llinsdorf, Switzerland) were approximately 10 weeks of age
(254 to 292 g) at the start of dosing. Clinical observations, body weight and food
consumption determinations, clinical pathology (hematology and clinical chemistry) evaluations, and gross pathology examinations without organ weight determinations (due to the premature sacrifice of all treated groups) were performed on all groups. Microscopic examinations were conducted on all gross
lesions and on a limited standard list of organs and tissues from animals assigned
to the control and low-dose groups. At day 1 and day 8 blood samples were
collected for toxicokinetic analyses.

RESULTS
Inhibitory activity (Ki): QN254 is a potent inhibitor of both
the WT and quadruple mutant plasmodium DHFR enzymes.
QN254 was compared to the antimalarials PM and cycloguanil
(inactive on QM PfDHFR), as well as the triazine WR99210, a
potent inhibitor of all mutant PfDHFRs, including QM
PfDHFR (20). The results of these experiments are summarized in Table 1. Consistent with previously published results
showing that QN254 is active against the highly PM-resistant
strain V1S, our results show that QN254 displays binding affinities comparable to WR99210 against both WT PfDHFR
(Ki 0.39 0.05 nM) and QM PfDHFR (Ki 0.58 0.06
nM). It is also worth noting that QN254 appears to show a
slightly better selectivity than WR99210, as shown by the

higher human/plasmodium Ki ratio (26 for QN254 versus 15

for WR99210).
WR99210 and QN254 have similar binding modes on QM
PfDHFR. To determine the structural features important for
QN254 binding to PfDHFR, we determined the crystal structure of a QN254/QM PfDHFR-TS (V1S) complex at 2.7-
resolution (see Table S1 in the supplemental material). Figure
1 shows the structure of this complex and shows the key interactions required for QN254 binding. QN254 makes direct interactions with several key residues in the DHFR active site, as
well as interactions via a water-mediated hydrogen bond. Compared to WR99210 binding (56), the diamino-quinazoline scaffold of QN254 hydrogen bonds with the side chain of Asp54
and gains an additional H-bond with Thr185 to replace the loss
of an H-bond with Tyr170 presumably due to the larger nature
of the quinazoline ring (Fig. 1A and B). Extensive hydrogen
bonds between the enzyme carbonyl backbones at Ile14, Cys15,
Leu164, and the diamino-quinazoline ring were established as
observed in the WR99210 structure. Interactions that increase
the binding of QN254 over WR99210 include a water-mediated hydrogen bond between the N-6 of the quinazoline ring
with both the side chain of Asn108 and the C-2 hydroxyl of the
nicotinamide adenosine dinucleotide phosphate (NADPH) ribose (Fig. 1A). In the WR99210 structure, a water molecule
also mediates a hydrogen bond between Ser111 and the ribose
of NADPH (Fig. 1B), but in this case, only enhances the
interaction with NADPH (56). QN254 makes additional van
der Waals contacts and - interactions with Leu46, Met55,
and Phe58 side chains. The phenyl substituent of both inhibitors makes similar van der Waals interactions with Ile112,
Pro113, Phe116, and Leu119. Consistent with the biochemical
data reported above, comparison of the QN254/QM PfDHFR
and WR99210/QM PfDHFR complexes (Fig. 1C) shows that
both compounds overlap extensively within the active site of
the QM PfDHFR enzyme. It appears that, in both cases, the
flexibility of the linker region between the diamino-quinazoline
or diamino-dihydrotriazine moieties, as well as the lipophilicity
of the phenyl ring are critically important for binding to the
QM PfDHFR. Moreover, QN254 takes advantage of the mutation at Asn108 by making an additional interaction via a
water molecule (Fig. 1A, 6-N. . .O, 3.22 )absent in the
WR99210/V1S structurethat presumably contributes to its
better affinity for the QM enzyme compared to WR99210.
QN254 shows potent antimalarial activity against P. falciparum PM drug-resistant clinical isolates with various PfDHFR
genotypes. To further demonstrate that QN254 is active
against P. falciparum even in areas of widespread PM drug

2606

NZILA ET AL.

ANTIMICROB. AGENTS CHEMOTHER.

published results obtained with QN254 (19). As expected, PM

was found to be largely inactive on these clinical isolates, with
a median IC50 of 733.26 nM. In agreement with these data, and
as discussed in our previous report, no WT isolates were found,
and more than 72% of the tested isolates carried triple mutations in Pfdhfr (S108N, N51I, and C59R). Interestingly, one
isolate carried the four mutations (S108N, N51I, C59R, and
I164L), mutations present in the V1S form (19).
In sharp contrast to the results obtained with PM, QN254
was potent against all isolates, with a median IC50 of 9.55 nM.
The median IC50s of QN254 against double mutants (S108N/
N51I or S108N/C59R) and triple mutants were 4.48 and 11.66
nM, respectively, and this difference was not statistically significant (Table 2). The quadruple mutant isolate had an IC50 of
7.61 nM. Collectively, these data clearly show that QN254 is
active on clinically relevant P. falciparum isolates with a wide
range of mutations in Pfdhfr (including quadruple mutations)
associated with significant PM drug resistance.
QN254 is orally bioavailable and displays slow but complete
absorption as well as a long half-life in rodents. To determine
the PK properties of QN254 in vivo, we measured the plasma
concentration-time profile upon p.o. and i.v. administration in
both mice and rats (Fig. 2). PK parameters are reported in
Table S2 in the supplemental material. In both rodent species,
QN254 displayed a high volume of distribution, greater than
the total body water (volume of distribution at steady state
[Vss] 2.85 and 4.7 liters/kg in mice and rats, respectively), and
the total systemic clearance was moderate to low at 57 and
30% of hepatic blood flow in mice and rats, respectively. Consistently, QN254 displayed an i.v. half-life of 0.8 h in mice and
a relatively longer half-life of 3.5 h rats. After oral administration, the elimination half-lives were around 6 and 9 h in mice
and rats, respectively. QN254 oral absorption was slow but
complete with a time to maximum concentration (Tmax) of 4 h
and apparent oral bioavailability of 100% in mice. These data
indicate that the low aqueous solubility of the compound (2
mg/liter at pH 6.8) does not limit its absorption in rodents. In
rats, QN254 absorption is similarly slow, and the relative oral
bioavailability is 63%. Notably, the maximum plasma concentrations (Cmax) values were about 5.1 and 2.8 M in mice and
rats, respectively, which are concentrations several hundredfold above the P. falciparum IC50.
QN254 is orally efficacious in the P. berghei-infected malaria
mouse model. We tested the efficacy of QN254 in the P. berghei

FIG. 1. Cocrystal structure of dihydrofolate reductase quadruple

mutant (mutations at codons 108, 51, 59, and 164) showing H-bond
interactions with QN254 (A) and WR99210 (B). Both complexes are
superposed in panel C.

resistance, we determined the in vitro activity of QN254 against

27 clinical isolates collected in Kilifi (Kenya) between 2006 and
2008. These isolates were part of a larger study in which we
measured the activity of standard antifolate drugs (including
PM) (19). For comparison purposes, in Table 2, we report
again the data pertaining to PM along with the previously

TABLE 2. Relationship between PM and QN254 median inhibitory

concentrations that kill 50% of parasitemia (IC50) and dihydrofolate
reductase (dhfr) genotypes of clinical isolatesa
IC50 (nM)c

DHFR genotypeb

Double mutant*
Triple mutant*
Quadruple mutant
a

PM

QN254

371 (8)
778.28 (24)
3,690.79 (1)

4.48 (6)
11.66 (20)
7.61 (1)

No WT dhfr isolate was identified.

The double mutants are S108N and N51I or S108N and C59R; the triple
mutants are S108, N51I and C59R; and the quadruple mutants are S108N, N51,
C59R, and I164L. *, None of the tested differences between double and triple
mutants were significant at P 0.05 (Kruskal-Wallis test).
c
The number of isolates is given in parentheses. Data pertaining to PM were
already published elsewhere (38).
b

VOL. 54, 2010

EVALUATION OF QN254 AS AN ANTIMALARIAL DRUG CANDIDATE

FIG. 2. PK profile of QN254 upon i.v. and p.o. administration.

QN254 was administered p.o. at 25 mg/kg (}) and i.v. at 5 mg/kg ()
to mice (A) and rats (B). Plasma concentrations were measured over
time and are indicated in ng/ml the standard deviations (n 3).
Note that the timescale is different in panels A and B. Drug levels for
the last time points in profile A were detectable but below the lower
limit of quantification (12 ng/ml).

malaria mouse model, a reference animal model (12). Upon

oral administration of single doses at 10, 15, 30, 60, and 100
mg/kg, QN254 showed a dose-dependent parasitemia reduction, 59 and 73% for 10- and 15-mg/kg doses, respectively;
99.99% for all other doses (Table 3). The efficacious doses of
QN254 required to inhibit the growth of 50% (ED50) and 90%
parasitemia (ED90) were 7.2 and 12.4 mg/kg, respectively, values that are comparable to those of artesunate (5.9 and 20.5
mg/kg) but slightly higher than those of mefloquine (3.8 and
5.2 mg/kg) and chloroquine (1.9 and 4.2 mg/kg) (52).
These results are consistent with the good PK data by the
oral route reported above; however, one should bear in mind
that the PK analysis was done in naive mice, and one cannot
rule out that the PK parameters in infected mice might be
different. Mouse survival was significantly increased to 8.1 days
with a single dose treatment at 30 mg/kg and increased to 12
days at 60 mg/kg. At 100 mg/kg, QN254 displayed activity
superior to the currently marketed antimalarial drugs artesunate and chloroquine, with parasitemia reduction 99.99%
and an average mouse survival prolongation of 23.4 days. In
addition, 50% of mice were cured with a single dose of 100
mg/kg QN254, whereas no mice were cured with a similar drug
treatment regimen for all three standard antimalarial drugs.
At three daily oral doses of 30 mg/kg, the cure rate was
improved to 80% with a mouse survival prolongation of about
28.4 days. We found some dose-limiting toxicity at three doses
of 60 mg/kg and three doses of 100 mg/kg since six of the ten
treated mice died at around day 10, despite being free of
parasites at the time. The four surviving mice were cured and
free of parasites at day 30.

2607

Collectively, our data show that QN254 displays potent in

vivo oral activity in the P. berghei mouse model and cured
infected mice upon administration of three daily oral doses as
low as 30 mg/kg. However, three-day dosing at higher doses
(60 mg/kg) led to toxicity and death in some animals. These
results indicate that the therapeutic window of QN254 might
be too narrow.
QN254 displays severe toxicity in rats and has a small
therapeutic window. In an exploratory 2-week rat toxicology
study we found that QN254 was not tolerated upon repeated
oral administration of daily dose of 50 mg/kg. In the present
study, all animals had to be euthanized by day eight, because of
serious adverse effects, body weight loss, decreased food consumption, and other clinical signs (soft feces, piloerection, and
reduced motor activity).
In all rats sacrificed preterm, histopathological analysis revealed marked gastrointestinal tract and bone marrow toxicity.
The major findings included degenerative/regenerative, atrophic and inflammatory changes in the gastric tract (mostly in
the small intestine and cecal mucosa) and massive bone marrow atrophy. The affected tissues being very proliferative, this
type of toxicity is consistent with on-target effects through
sustained inhibition of DHFR and inhibition of cell proliferation, as has been previously reported for other DHFR inhibitors (2, 54).
Toxicokinetics data generated in the course of the present
study showed that in rats, upon administration of a 50-mg/kg

TABLE 3. QN254 antimalarial activity in the P. berghei murine

malaria modela
Dosing regimen
(agent and dose
mg/kg)b

Parasitemia
reduction (%)

Avg mouse
survival (days)

Cure
rate
(%)

Toxicity
rate
(%)c

QN254
1 10
1 15
1 30
1 60
1 100
3 30
3 60
3 100

59
73
99.99
99.99
99.99
99.99
99.99
99.99

6.0
6.0
8.1
12.0
23.4
28.4
NAd
NA

0
0
0
0
50
80
60
20

0
0
0
0
0
0
40*
80*

Artesunate
1 100
3 30

98
98.7

7.0
12.2

0
0

0
0

Chloroquine
1 100
3 30

99.6
99.8

12.5
14.3

0
0

0
0

Mefloquine
1 100
3 30

89
98.8

25.5
22.1

0
0

0
0

a
Results represent the percent inhibition of parasitemia compared to untreated controls. QN254 was prepared in 0.5% carboxymethyl cellulose. The
mean survival of control animals was 5.6 to 6.2 days.
b
The solution formulation for artesunate, chloroquine, and mefloquine consisted of 10% ethanol, 30% PEG 400 (polyethylene glycol 400), and 60% of a
10% vitamin-ETPGS solution.
c
*, Mice died around day 10 and were parasite-free. There were 10 mice per
test group except for the 3 treatment with QN254 because of ethical reasons
due to the observed toxicity.
d
NA, not available.

2608

NZILA ET AL.

dose of QN254, daily plasma exposure at day 1 (AUC024

27,600 ng h/ml) is only about twice the plasma exposure level
reached at the efficacious dose in mice (AUC024 16,990
ng h/ml at an ED99 of 26.5 mg/kg). Thus, the QN254 therapeutic window is smaller than 2.
DISCUSSION
We have previously shown that QN254 is active in vitro
against the P. falciparum strain (V1S) carrying a copy of the
QM-Pfdhfr gene. Here, we provide data that the diaminoquinazoline DHFR inhibitor QN254 (i) binds to and inhibits
the QM PfDHFR enzyme; (ii) displays potent antimalarial
activity against antifolate drug-resistant P. falciparum clinical
isolates with various DHFR genotypes, including QM-Pfdhfr;
(iii) shows PK properties compatible with oral dosing; and (iv)
displays excellent in vivo therapeutic efficacy in the P. berghei
malaria mouse model.
Because antifolate drugs such as PM and proguanil have lost
their efficacy due to resistance, it is crucial for any new molecule targeting this enzyme to show activity against all PMresistant DHFR mutant forms. In an earlier study, we assessed
the in vitro activity of WR99210 against the same clinical isolates tested in this publication (mean IC50 of about 0.72 nM
[19]). Thus, compared to QN254, WR99210 is about 10 times
more potent against parasite despite having a slightly lower
binding affinity to PfDHFR than QN254. It remains to be
determined whether better diffusion across membranes of parasitized red blood cells and/or an additional off-target effect
could explain the higher WR99210 cellular potency.
Using a functional/biochemical assay and crystallographic
structural data, we provide further evidence that QN254 binds
to and inhibits Plasmodium WT and QM DHFR enzymes
efficiently. The resolution of the three-dimensional structure of
WT and QM PfDHFRwith either PM or WR99210 bound to
its active siteprovided structural insights into DHFR PM
resistance mechanisms, as well as some understanding of the
structural features allowing WR99210 to retain affinity for QM
PfDHFR (55, 56). Our crystallographic data, describing the
structure of a ternary complex of QN254 bound to QM
PfDHFR, show that QN254 binds to its target in a manner
similar to WR99210. Previous X-ray crystallographic studies
(5) with a compound closely related to QN254 and the Pneumocystis carinii DHFR enzyme suggested that Phe69 (P. carinii
numbering) or Phe116 (P. falciparum numbering) was engaged
in close hydrophobic contact with the 5 methoxy group on the
phenyl ring of the lipophilic tail. The authors of that study also
speculated that this interaction may contribute to the relative
selectivity toward P. carinii (and possibly P. falciparum) since
the conserved phenylalanine was not present in the mammalian DHFR enzymes. Careful analysis of our cocrystal QN254/
PfDHFR structure failed to reveal such an interaction and in
fact we could only identify weak van der Waals interactions
between the lipophilic tail and the hydrophobic cavity present
in the active site of QM PfDHFR. The structure-activity relationship (SAR) data for the class of quinazoline compounds
are not very clear and are currently limited to what is described
in previous publications (811, 38, 39).
Consistent with our structural data demonstrating the absence of specific interactions between the phenyl ring tail and

ANTIMICROB. AGENTS CHEMOTHER.

PfDHFR, the available SAR data show that a large number of

modifications of the lipophilic tails are tolerated. With respect
to selectivity, our data do not provide any obvious structural or
molecular explanation to rationalize the relative selectivity of
QN254 for P. falciparum versus the human enzyme.
For more than 30 years, it has been known that QN derivatives inhibit the growth and proliferation of eukaryotic parasites such as Toxoplasma, Pneumocystis, Trypanosoma, and
Plasmodium (8, 10, 11, 23, 36, 38, 45, 46). In fact, an extensive
series of quinazolines have previously proved to be efficacious
in rodent and simian malaria models (4144). The most advanced candidate of this series, WR158122, entered clinical
development as a candidate antimalarial drug but was later
abandoned because of its relatively poor oral efficacy in humans (43).
After oral administration QN254 is well absorbed, displays a
long half-life and an excellent oral bioavailability in rodents, as
well as in larger species such as dog (data not shown). Although PAMPA and Caco-2 in vitro permeability data were
predictive of the good absorption and bioavailability, our in
vitro metabolic stability data predicted that QN254 would be
subject to rapid hepatic clearance in all preclinical species, as
well as in humans (data not shown). Nonetheless, in vivo
QN254 showed a low (rats and dogs) to moderate (mouse)
systemic clearance in all preclinical species. We cannot explain
the discrepancy between in vitro and in vivo hepatic clearance,
and further studies would be required to determine the main
clearance mechanism in vivo.
Consistent with our PK results, QN254 displays good efficacy
in the P. berghei mouse model through the oral route. However, in P. berghei-infected mice, we have found evidence of
toxicity upon repeated daily dosing for 3 days at doses as low
as 60 mg/kg and, indeed, further rat toxicology studies showed
that QN254 does not possess an adequate therapeutic window.
At this juncture we cannot unambiguously establish whether
the observed toxicity is due to on- or off-target effects; as an
example, we have evidence that QN254 presents some inherent
risk of cardiotoxicity because of its ability to bind and inhibit
the hERG channel in vitro (hERG IC50 1.4 M). Nonetheless, the rat histopathological data showing that a highly proliferative tissue such as the bone marrow is massively affected
is consistent with on-target toxicity and sustained inhibition of
folates metabolism through DHFR inhibition (50, 54). Thus,
despite its relative selectivity toward plasmodium DHFR enzyme in vitro, QN254 appears to potently inhibit mammalian
(rat) DHFR in vivo. Based on the exposure reached at the
efficacious dose and the exposure reached in rats upon dosing
at 50 mg/kg in the toxicology study, we estimated that the
therapeutic window is less than two. A narrow therapeutic
window is indeed a hallmark of almost all antifolates and has
historically been a significant hurdle to the development of
new therapies based on DHFR inhibition. PM, for example,
displays an acute LD50 of 130 mg/kg in mice (50). Mitigating
this toxicity risk is the fact that antifolates such as PM are
commonly used in combination at low doses (5 mg/kg in
humans). Because of the severe toxicity observed in rats, the
Novartis Institute for Tropical Diseases is no longer pursuing
the development of QN254. Nevertheless, further research
into the feasibility of strategiese.g., a prodrug approach,
supplementation with folate derivatives such as 5-methyl-THF

VOL. 54, 2010

EVALUATION OF QN254 AS AN ANTIMALARIAL DRUG CANDIDATE

(27), or a combination with antimalarials showing synergistic

effects with DHFR inhibitorsaiming to increase the therapeutic window of QN254 may be warranted.

17.

ACKNOWLEDGMENTS
We thank the director of the Kenya Medical Research Institute for
permission to publish these data.
S.K. is an international research scholar of Howard Hughes Medical Institute. The Kenya Medical Research Institute received support from the EU Commission under Framework 6 as part of the
AntiMal Integrated Project 018834, the European and Developing
Countries Clinical Trials Partnership, and the Wellcome Trust
WT077092. BIOTEC was supported by grants from Thailand-TDR
(T-2) and the Medicines for Malaria Venture. Novartis Institute for
Tropical Diseases and the Swiss Tropical Institute receive funding
from a joint grant of the Medicines for Malaria Venture and the
Wellcome Trust (WT078285).

18.

19.

20.

21.
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