Preclinical evaluation of antifolate QN254 (5-chloro-N*6*-(2,5-dimethoxy-benzyl)-1
quinazoline-2,4,6-triamine) as antimalarial drug candidate 2
3
Alexis Nzila1,2,*, Matthias Rottmann3, Penchit Chitnumsub4, Stevens M. Kiara1, Sumalee 4
Kamchonwongpaisan4, Cherdsak Maneeruttanarungroj4, Supannee Taweechai4, Bryan K.S. 5
Yeung5, Anne Goh5, Suresh B. Lakshminarayana5, Bin Zou5, Josephine Wong5, Ngai Ling 6
Ma5, Weaver Margaret6, Thomas H. Keller5, Veronique Dartois5, Sergio Wittlin3, Reto Brun3, 7
Yongyuth Yuthavong4 and Thierry T. Diagana5 8
9
10
1Kenya Medical Research Institute (KEMRI)/Wellcome Trust Collaborative Research 11
Program, PO Box 230, 80108, Kilifi, Kenya; 2University of Oxford, Nuffield Department of 12
Medicine, John Radcliffe Hospital, Oxford, UK; 3Swiss Tropical Institute (STI) Parasite 13
Chemotherapy, Socinstrasse 57, PO Box, CH-4002 Basel, Switzerland; 4National Centre for 14
Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, NSTDA, 15
Pathumthani 12120, Thailand; 5Novartis Institute for Tropical Diseases (NITD), 10 Biopolis 16
Road #05-01 Chromos, 138670 Singapore; 6Novartis Institute for Biomedical Research 17
(NIBR), 250 Massachusetts Ave, Cambridge, MA 02139, USA 18
19
*Corresponding author: Dr Alexis Nzila, Kenya Medical Research Institute 20
(KEMRI)/Wellcome Trust Collaborative Research Program, PO Box 230, 80108 Kilifi, Kenya. 21
Tel: +254-417-522-535; fax: +254-417-522-290; e-mail: [email protected] 22
23
Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Antimicrob. Agents Chemother. doi:10.1128/AAC.01526-09 AAC Accepts, published online ahead of print on 29 March 2010
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
2
1 Abstract 24
Drug resistance against dihydrofolate reductase (DHFR) inhibitors – such as pyrimethamine 25
(PM) – has now spread to almost all malaria endemic regions, rendering antifolate-based 26
malaria treatments highly ineffective. We have previously shown that the di-amino quinazoline 27
QN254 is active against the highly pyrimethamine-resistant Plasmodium falciparum V1S 28
strain, suggesting that QN254 could be used to treat malaria in regions with a high prevalence 29
of antifolate resistance. Here, we further demonstrate that QN254 is highly active against 30
Plasmodium falciparum clinical isolates, displaying various levels of antifolate drug resistance, 31
and we provide biochemical and structural evidence that QN254 binds and inhibits the function 32
of both the wild-type and quadruple-mutant form (V1S) of the DHFR enzyme. In addition, we 33
have assessed QN254 oral bioavailability, efficacy and safety in vivo. The compound displays 34
favorable pharmacokinetic properties following oral administration in rodents. The drug was 35
remarkably efficacious against Plasmodium berghei and could fully cure infected mice with 36
three daily oral doses of 30 mg/kg. In the course of these efficacy studies, we have uncovered 37
some dose limiting toxicity at higher doses that was confirmed in rats. Thus, despite its relative 38
in vitro selectivity toward the Plasmodium DHFR enzyme, QN254 does not show the adequate 39
therapeutic index to justify its further development as a single agent. 40
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
3
41
2 Introduction 42
Malaria control is a global public health priority that has been hampered by the rapid 43
development and spread of resistance against antimalarials. As a consequence, the WHO 44
recommends the use of artemisinin-containing combination therapies (ACTs) as first-line 45
treatment for malaria. Although ACTs are designed to reduce the chance of artemisinin drug 46
resistance development, there are considerable concerns that this may already have occurred. 47
For instance, there is now mounting evidence that the efficacy of artemisinin derivatives is 48
reduced in South-East Asia, where artemisinin derivatives have been used for a long time as 49
monotherapies (7, 28, 53). This is a cause of concern since the spread of artemisinin resistance 50
will compromise the usefulness of ACTs globally. Thus there is an urgent need to discover and 51
develop new alternative drugs. 52
For several decades, dihydrofolate reductase (DHFR) has been targeted with different classes 53
of chemical entities for the development of new therapies for a broad range of therapeutic 54
indications including several parasitic diseases (13). DHFR catalyzes the reduction of 55
dihydrofolate (DHF) to tetrahydrofolate (THF), which is an essential cofactor in the 56
biosynthesis of deoxythymidylate monophosphate (dTMP), a metabolite essential to DNA 57
synthesis and cell replication. 58
Pyrimethamine (PM) is a potent inhibitor of the Plasmodium DHFR enzyme and this 59
compound has been widely used in combination with the dihydropteroate synthetase (DHPS) 60
inhibitor sulfadoxine. Unfortunately, pyrimethamine resistance is now common, rendering this 61
drug ineffective (30). One of the documented mechanisms of antifolate resistance is through 62
the mutation of the target itself – the pfdhfr-ts gene, encoding for a bi-functional enzyme which 63
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
4
possesses both DHFR and thymidylate synthase (TS) activities carried by two distinct sub-64
domains (29). Pyrimethamine resistance is associated with the mutation of the amino acid Ser 65
to Asn at codon 108 of DHFR (S108N). Ancillary mutations of N51I and C59R are associated 66
with an increase in resistance, and the presence of the mutation I164L results in an even higher 67
level of PM resistance (14, 47). The presence of these mutations significantly decreases the 68
sensitivity of the PfDHFR enzyme to pyrimethamine inhibition in a biochemical assay (48). 69
The antifolate triazine WR99210 (3, 22) is potent against Plasmodium falciparum bearing 70
quadruple mutations of DHFR at S108N, N51I, C59R, and I164L (QM PfDHFR) (20). 71
However, WR99210 has shown limited efficacy in vivo due to poor oral bioavailability and 72
displayed some gastrointestinal toxicity. Attempts have been made to circumvent these issues 73
and a pro-drug form of WR99210 known as PS-15 has been shown to be orally active (2). The 74
resolution of the three-dimensional structure of wild-type (WT) and QM PfDHFR – with either 75
PM or WR99210 bound to its active site – provided structural insights into DHFR 76
pyrimethamine resistance mechanisms as well as some understanding of the structural features 77
of WR99210 that allow this compound to retain affinity for QM PfDHFR (55, 56). 78
The quinazoline (QN) pharmacophore has been successfully employed to design drugs for the 79
treatment of cancer and other human diseases. For example, the DHFR inhibitor, trimetrexate 80
(5-methyl-6-[(3,4,5-trimethoxy-phenylamino)-methyl]-quinazoline-2,4-diamine) has been 81
developed to treat various cancers in human patients (15, 32, 33). In a previous study, a series 82
of quinazoline derivatives was tested against the highly pyrimethamine-resistant Plasmodium 83
falciparum strain (V1S) and the DHFR inhibitor 2,4-diamino-5-chloro-6-[N-(2,5-84
dimethoxybenzyl)-amino]quinazoline (or QN254 in this paper and compound 1 in (34)) was 85
found to have potent activity – with an IC50 value (inhibitory concentration that reduces 86
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
5
parasite growth by 50% in vitro) of 9 nM (34). Considering the increasingly widespread 87
pyrimethamine drug resistance, we set out to perform the experiments described in this paper 88
with the goal of further assessing the potential of QN254 as a candidate antimalarial to replace 89
the failing pyrimethamine. Collectively, our data demonstrate that QN254 (i) binds and inhibits 90
the QM PfDHFR enzyme, (ii) is active on drug-resistant clinical isolates and (iii) displays 91
pharmacological properties compatible with an oral antimalarial drug candidate. However, 92
preliminary toxicological findings indicate that QN254 does not show a therapeutic window 93
sufficiently large to warrant its progression to the next development stage. 94
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
6
3 Material and Methods 95
Drugs and reagents 96
Pyrimethamine (PM) and chloroquine (CQ) used as standard drugs for evaluation on clinical 97
isolates were purchased from Sigma (Cole, UK). WR99210 was a gift from Professor Steve 98
Ward (Liverpool Tropical School, UK). In the Ki determination, cycloguanil (CGL) and 99
WR99210 were gifts from Professor Tirayut Vilaivan (Chulalongkorn University, Thailand). 100
Standard drugs for in vivo efficacy testing were obtained from Mepha Ltd, Switzerland 101
(artesunate [ART]), Sigma, USA (chloroquine diphosphate) and F. Hoffmann-LaRoche Ltd, 102
Switzerland (mefloquine hydrochloride). QN254 was prepared as described elsewhere (39). 103
DHFR biochemical assay and X-ray structure determination 104
Ki determination: Recombinant PfDHFR enzymes of P. falciparum and human were prepared 105
from E. coli BL21(DE3) bearing pET17b expression plasmids of P. falciparum wild-type (WT) 106
and quadruple mutant (S108N, N51I, C59R, I164L) [QM pfdhfr] (17), and human dhfr (hdhfr) 107
(18), using a methotrexate column as described previously (51). Binding affinities (Ki values) 108
of QN254 and standard antifolate antimalarials, cycloguanil, pyrimethamine and WR99210 109
were determined as described elsewhere (51). 110
X-Ray structure determination 111
Preparation of recombinant PfDHFR-TS bifunctional enzymes was carried out as previously 112
reported (4). Crystals were flash-frozen in liquid nitrogen by dipping for 10 s in a 113
corresponding crystallization solution containing 20% glycerol as a cryoprotectant. X-ray 114
diffraction data were collected under a cold nitrogen stream (100 K) at a wavelength of 1.54 Å 115
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
7
on an FR591 rotating anode X-ray generator equipped with a nonius KappaCCD detector. Data 116
were processed using DENZO and SCALEPACK in the HKL2000 suite (35). Refinement was 117
performed using a 2.1 Å 1J3K crystal structure as a template in both CNS (1) and REFMAC5 118
(26) along with model building in program O (16) and model validation in PROCHECK (24, 119
25) and Moleman2 (21). Figures were prepared with PyMOL (6). 120
Plasmodium strains and culturing methods 121
3.1.1 Parasite strains and isolates 122
We analyzed the in vitro activity of QN254 against clinical isolates from Kenya with different 123
dhfr genotypes. These clinical isolates were collected in Kilifi between 2006 and 2008 as part 124
of the malaria studies, and were in vitro adapted for long-term culture as described elsewhere 125
(40). 126
3.1.2 In vitro culture and chemosensitivity test 127
P. falciparum cultures were carried out in RPMI 1640 (GIBCO BRL, UK) medium 128
supplemented with 10% (v/v) normal human serum, 25 mM bicarbonate, 2 mM glutamine, 25 129
mM HEPES buffer, physiological concentrations of para-aminobenzoic acid (5 nM) and folic 130
acid (23 nM). Antimalarial activity was measured in the presence of varying concentrations of 131
each compound using radioisotopic incorporation (49). Results were expressed as the drug 132
concentration required for 50% inhibition (IC50) of [3H]hypoxanthine incorporation into 133
parasite nucleic acid, using non-linear regression analysis of the dose–response curve. 134
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
8
3.1.3 Parasite genotyping of dhfr 135
After in vitro adaptation of parasites, infected blood samples were spotted onto filter paper and 136
stored. Parasite genomic material from these filter papers was prepared using the methanol 137
procedure and point mutations at codons 108, 51, 59 and 164 of dhfr were analyzed by PCR 138
and restriction enzyme digestion (PCR-RFLP) as described elsewhere (31). Statistical analyses 139
were carried out using Stata, version 9 (StataCorp, College Station, TX, USA), using the 140
Kruskal–Wallis non-parametric test for comparison of medians. The level of significance was 141
set at p<0.05. 142
In vivo pharmacokinetic studies 143
The Institutional Animal Care and Use Committee (IACUC) of Novartis Institute for Tropical 144
Diseases, registered with the Agri-Food and Veterinary Authority (AVA), Government of 145
Singapore, approved all animal experimental protocols. QN254 was formulated at a 146
concentration of 2.5 mg/ml for a dose of 25 mg/kg given orally (p.o.) and at 1 mg/ml 147
concentration for a dose of 5 mg/kg given intravenously (i.v.).The solution formulation for i.v. 148
dosing contained 10% NMP (n-methyl pyrrolidone), 30% PEG 400 and 60% of 10% Vitamin 149
E-TPGS. The suspension formulation for p.o dosing was 0.5% CMC (carboxymethyl 150
cellulose). For PK studies, blood samples were collected at several time points following p.o. 151
and i.v. dosing in mice and rats. 152
3.1.4 Extraction and LCMS analysis of QN254 153
Plasma samples were extracted with acidified acetonitrile for QN254 using an extractant to 154
plasma ratio of 8:1. Analyte quantification was performed by LC/MS/MS. Liquid 155
chromatography was performed using an Agilent 1100 HPLC system (Santa Clara, CA, USA), 156
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
9
with an Agilent Zorbax XDB Phenyl (3.5µ, 4.6 × 75 mm) column at an oven temperature of 157
35°C, coupled with an API3200 triple quadruple mass spectrometer (Applied Biosystems, 158
Foster City, CA, USA). Instrument control and data acquisition were performed using Analyst 159
1.4.2 software (Applied Biosystems). The mobile phases used were A: water–acetic acid 160
(99.8:0.2, v/v) and B: acetonitrile–acetic acid (99.8:0.2, v/v), using a gradient, with flow rate of 161
1.0 ml/min and run time of 5 min. Under these conditions, the analyte retention time was 2.9 162
min. Compound detection on the mass spectrometer was performed in electrospray positive 163
ionization mode and using multiple reaction monitoring (MRM) for specificity (transitions 164
360.3/209.2 and 360.3/174.2) together with their optimized MS parameters. Analysis of study 165
samples was performed on different days using an identical method with a ten-point calibration 166
curve spanning three orders of magnitude in compound concentration. The lower and upper 167
limits of quantification were 12 ng/ml and 7500ng/mL respectively. Intra-day variability was 168
established with triplicate quality control samples at three concentration levels. Relative 169
standard deviation was under 15%. Inter-day variability is not reported as calibration curves 170
were re-prepared and reanalyzed with every set of study samples and results accepted if inter-171
day variability was found to be under 15%. 172
3.1.5 Pharmacokinetic data analysis 173
The mean value from three animals at each time point was plotted against time to give the 174
plasma concentration–time profile. Pharmacokinetic parameters were determined using 175
WinNonlin Professional, version 5.0.1 (Pharsight, CA, USA), by non-compartmental modeling 176
using software model 200 for oral dosing and model 201 for intravenous dosing. The oral 177
bioavailability (F) was calculated as the ratio between the area under the curve (AUC) 178
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
10
following oral administration and the AUC following intravenous administration corrected for 179
dose (F = AUC p.o. × dose i.v./AUC i.v. × dose p.o.). 180
In vivo antimalarial efficacy studies 181
All in vivo efficacy studies were carried out at the Swiss Tropical Institute, adhering to the 182
regulations of the veterinary authorities of Basel-Stadt. The murine P. berghei ANKA was 183
used as previously described (37). Experimental and control groups of 5 female NMRI mice 184
(20–22 g) were infected intravenously with 1 × 108 parasitized erythrocytes/ml, in a volume of 185
0.2 ml (from a fresh mouse donor). In untreated control mice, parasitemia rises regularly to 186
~30% by day 3 post-infection and causes death of the animals between day 5 and day 7 post-187
infection. 188
Experimental compounds were prepared in 0.5% carboxymethyl cellulose (CMC) and 189
administered orally. Comparators (Artesunate, Chloroquine and Mefloquine) were prepared in 190
ETPGS vehicle (10% EtOH, 30% PEG400 and 60% of a 10% Vitamin ETPGS solution) and 191
administered orally. Parasitemia was determined microscopically. Activity was calculated as 192
the difference between parasitemia for the control and treated groups expressed as a per cent 193
relative to the control group. The survival time was also recorded up to 30 days after infection. 194
A compound was considered curative if the animal survived to day 30 after infection with no 195
detectable parasites. The ED50 and ED90 values were assessed at day 3 by plotting the dose as a 196
function of parasitemia, using non-linear fitting with the Microcal Origin Statistical Program 197
(OriginLab, Northampton, MA, USA). All data reported are based on n≥10 mice, except for 198
the three times treatment with QN254 which, because of the observed toxicity, were performed 199
only once with a cohort of 5 mice. 200
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
11
Rat toxicological study 201
QN254 was suspended in 10 % of a 1% (v/v) aqueous Solutol HS15 solution and 90% of a 202
0.5 % aqueous Methylcellulose M0555 solution and administered to groups of 5 male rats 203
at daily oral (by gavage) doses of 50, 150 or 500 mg/kg/day. Animals (Wistar rats, Harlan 204
Laboratories Ltd., Füllinsdorf, Switzerland) were approximately 10 weeks of age (254 to 205
292 g) at the start of dosing. Clinical observations, body weight and food consumption 206
determinations, clinical pathology (hematology and clinical chemistry) evaluations, gross 207
pathology examinations without organ weight determinations (due to premature sacrifice 208
of all treated groups) were performed on all groups. Microscopic examinations were 209
conducted on all gross lesions and on a limited standard list of organs and tissues from 210
animals assigned to the control and low-dose groups. At day 1 and day 8 blood samples 211
were collected for toxicokinetic analyses. 212
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
12
4 Results 213
Inhibitory activity (Ki): QN254 is a potent inhibitor of both the wild-type and quadruple 214
mutant plasmodium DHFR enzymes 215
QN254 was compared to the antimalarials pyrimethamine (PM) and cycloguanil (CGL) – 216
inactive on QM PfDHFR – as well as the triazine WR99210, a potent inhibitor of all mutant 217
PfDHFR, including QM PfDHFR (20). The results of these experiments are summarized in 218
Table 1. Consistent with previously published results showing that QN254 is active against the 219
highly pyrimethamine-resistant strain V1S, our results show that QN254 displays binding 220
affinities comparable to WR99210 against both WT PfDHFR (Ki= 0.39 ± 0.05 nM) and QM 221
PfDHFR (Ki=0.58 ± 0.06 nM). It is also worth noting that QN254 appears to show a slightly 222
better selectivity than WR99210 as shown by the higher human/plasmodium Ki ratio (26 for 223
QN254 vs 15 for WR99210). 224
WR99210 and QN254 have similar binding modes on QM PfDHFR 225
To determine the structural features important for QN254 binding to PfDHFR, we solved the 226
crystal structure of a QN254/QM PfDHFR-TS (V1S) complex at 2.7 Å resolution 227
(Supplementary Table S1). Figure 1 describes the structure of this complex and shows the key 228
interactions required for QN254 binding. QN254 makes direct interactions with several key 229
residues in the DHFR active site as well as interactions via a water-mediated hydrogen bond. 230
Compared to WR99210 binding (56), the diamino-quinazoline scaffold of QN254 hydrogen 231
bonds with the side chain of Asp54 and gains an additional H-bond with Thr185 to replace the 232
loss of an H-bond with Tyr170 presumably due to the larger nature of the quinazoline ring 233
(Figure 1A and 1B). Extensive hydrogen bonds between the enzyme carbonyl backbones at 234
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
13
Ile14, Cys15, Leu164 and the diamino-quinazoline ring were established as observed in the 235
WR99210 structure. Interactions which increase the binding of QN254 over WR99210 include 236
a water-mediated hydrogen bond between the N-6 of the quinazoline ring with both the side 237
chain of Asn108 and the C-2 hydroxyl of the nicotinamide adenosine dinucleotide phosphate 238
(NADPH) ribose (Figure 1A). In the WR99210 structure, a water molecule also mediates a 239
hydrogen bond between Ser111 and the ribose of NADPH (Figure 1B), but in this case, only 240
enhances the interaction with NADPH (56). QN254 makes additional van der Waals contacts 241
and π−π interactions with Leu46 and Met55 and Phe58 side chains. The phenyl substituent of 242
both inhibitors makes similar van der Waals interactions with Ile112, Pro113, Phe116 and 243
Leu119. Consistent with the biochemical data reported above, comparison of the QN254/QM 244
PfDHFR and WR99210/QM PfDHFR complexes (Figure 1C) shows that both compounds 245
overlap extensively within the active site of the QM PfDHFR enzyme. It appears that, in both 246
cases, the flexibility of the linker region between the diamino-quinazoline or diamino-247
dihydrotriazine moieties as well as the lipophilicity of the phenyl ring are critically important 248
for binding to the QM PfDHFR. Moreover, QN254 takes advantage of the mutation at Asn108 249
by making an additional interaction via a water molecule (Figure 1A: 6-N…O, 3.22 Å) – 250
absent in the WR99210/V1S structure – which presumably contributes to its better affinity for 251
the QM enzyme compared with WR99210. 252
QN254 shows potent antimalarial activity against P. falciparum pyrimethamine drug-253
resistant clinical isolates with various PfDHFR genotypes 254
To further demonstrate that QN254 is active against P. falciparum even in areas of widespread 255
PM drug resistance, we determined the in vitro activity of QN254 against 27 clinical isolates 256
collected in Kilifi (Kenya) between 2006 and 2008. These isolates were part of a larger study 257
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
14
in which we measured the activity of standard antifolate drugs (including PM) (19). For 258
comparison purposes, in Table 2, we report again the data pertaining to PM along with the 259
previously published results obtained with QN254 (19). As expected, PM was found to be 260
largely inactive on these clinical isolates, with a median IC50 of 733.26 nM. In agreement with 261
these data, and as discussed in our previous report, no wild-type isolates were found and more 262
than 72% of the tested isolates carried triple mutations in pfdhfr (S108N+N51I+C59R). 263
Interestingly, one isolate carried the four mutations (S108N+N51I+C59R+I164L), mutations 264
present in the V1S form (19). 265
In sharp contrast to the results obtained with PM, QN254 was potent against all isolates, with a 266
median IC50 value of 9.55 nM. The median IC50 values of QN254 against double mutants 267
(S108N+N51I or S108N+C59R) and triple mutants were 4.48 nM and 11.66 nM, respectively, 268
and this difference was not statistically significant (Table 2). The quadruple mutant isolate had 269
an IC50 of 7.61 nM. Collectively, these data clearly show that QN254 is active on clinically 270
relevant P. falciparum isolates with a wide range of mutations in pfdhfr (including quadruple 271
mutations) associated with significant PM drug resistance. 272
QN254 is orally bioavailable and displays slow but complete absorption as well as a long 273
half-life in rodents 274
To determine the pharmacokinetic (PK) properties of QN254 in vivo, we measured the plasma 275
concentration–time profile upon oral and intravenous administration in both mice and rats 276
(Figure 2). Pharmacokinetic parameters are reported in Supplementary Table S2. In both rodent 277
species, QN254 displayed a high volume of distribution, greater than total body water (volume 278
of distribution at steady state (Vss)=2.85 and 4.7 l/kg in mice and rats, respectively), and the 279
total systemic clearance was moderate to low at 57% and 30% of hepatic blood flow in mice 280
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
15
and rats, respectively. Consistently, QN254 displayed an intravenous (i.v.) half-life of 0.8 h in 281
mice and a relatively longer half-life of 3.5 h rats. Following oral administration, the 282
elimination half-life was around 6 h and 9 h in mice and rats, respectively. QN254 oral 283
absorption was slow but complete with a time to maximum concentration (Tmax) of 4 h and 284
apparent oral bioavailability of 100% in mice. These data indicate that the low aqueous 285
solubility of the compound (~2 mg/l at pH 6.8) does not limit its absorption in rodents. In rats, 286
QN254 absorption is similarly slow and the relative oral bioavailability is 63%. Notably 287
maximum plasma concentrations (Cmax) values were about 5.1 and 2.8 µM in mice and rats, 288
respectively, which are concentrations several hundred fold above the P. falciparum IC50 value. 289
QN254 is orally efficacious in the P. berghei infected malaria mouse model 290
We tested the efficacy of QN254 in the P. berghei malaria mouse model, a reference animal 291
model (12). Upon oral administration of single doses at 10, 15, 30, 60 and 100 mg/kg, QN254 292
showed a dose-dependent parasitemia reduction, 59% and 73% for 10 and 15 mg/kg doses, 293
respectively; >99.99% for all other doses (Table 3). The efficacious doses of QN254 required 294
to inhibit the growth of 50% (ED50) and 90% parasitemia (ED90) were 7.2 and 12.4 mg/kg, 295
respectively, values that are comparable to those of artesunate (5.9 and 20.5 mg/kg) but slightly 296
higher than mefloquine (3.8 and 5.2 mg/kg) and chloroquine (1.9 and 4.2 mg/kg) (52). 297
These results are consistent with the good pharmacokinetic (PK) data by the oral route reported 298
above, however one should bear in mind that the PK analysis was done in naive mice and one 299
cannot rule out that the PK parameters in infected mice might be different. Mouse survival was 300
significantly increased to 8.1 days with a single dose treatment at 30 mg/kg and to 12 days at 301
60 mg/kg. At 100 mg/kg, QN254 displayed activity superior to the currently marketed 302
antimalarial drugs artesunate and chloroquine, with parasitemia reduction >99.99% and an 303
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
16
average mouse survival prolongation of 23.4 days. In addition, 50% of mice were cured with a 304
single dose of 100 mg/kg QN254, while no mice were cured with a similar drug treatment 305
regimen for all three standard antimalarial drugs. 306
At three daily oral doses of 30 mg/kg, the cure rate was improved to 80% with a mouse 307
survival prolongation of about 28.4 days. We found some dose-limiting toxicity at 3 × 60 and 3 308
× 100 mg/kg as six out of the 10 treated mice died at around day 10, despite being free of 309
parasites at the time. The four surviving mice were cured and free of parasites at day 30. 310
Collectively, our data show that QN254 displays potent in vivo oral activity in the P. berghei 311
mouse model and cured infected mice upon administration of three daily oral doses as low as 312
30 mg/kg. However, three-day dosing at higher doses (≥ 60 mg/kg) led to toxicity and death in 313
some animals. These results indicate that the therapeutic window of QN254 might be too 314
narrow. 315
QN254 displays severe toxicity in rats and has a small therapeutic window 316
In an exploratory 2-week rat toxicology study we found that QN254 was not tolerated upon 317
repeated oral administration of daily dose ≥ 50 mg/kg. In this study, all animals had to be 318
euthanized by day eight, because of serious adverse effects, body weight loss, decreased food 319
consumption and other clinical signs (soft feces, piloerection, reduced motor activity). 320
In all rats sacrificed preterm, histopathological analysis revealed marked gastrointestinal tract 321
and bone marrow toxicity. The major findings included degenerative/regenerative, atrophic and 322
inflammatory changes in the gastric tract—mostly in the small intestine and cecal mucosa—as 323
well as massive bone marrow atrophy. The affected tissues being very proliferative, this type of 324
toxicity is consistent with on-target effects through sustained inhibition of DHFR and 325
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
17
inhibition of cell proliferation as it has been previously reported for other DHFR inhibitors (2, 326
54). 327
Toxicokinetics data generated in the course of this study showed that in rats, upon 328
administration of a 50 mg/kg dose of QN254, daily plasma exposure at day 1 (AUC0-24h= 329
27,600 ng*h/mL) is only about 2 times the plasma exposure level reached at the efficacious 330
dose in mice (AUC0-24h≈ 16,990 ng*h/mL at the ED99=26.5 mg/kg). Thus QN254 therapeutic 331
window is smaller than 2. 332
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
18
333
5 Discussion 334
We have previously shown that QN254 is active in vitro against the P. falciparum strain (V1S) 335
carrying a copy of the QM-pfdhfr gene. Here, we provide data that the diamino-quinazoline 336
DHFR inhibitor QN254 (i) binds to and inhibits the QM PfDHFR enzyme, (ii) displays potent 337
antimalarial activity against antifolate drug-resistant P. falciparum clinical isolates with 338
various DHFR genotypes, including QM-pfdhfr, (iii) shows pharmacokinetic properties 339
compatible with oral dosing and (iv) displays excellent in vivo therapeutic efficacy in the P. 340
berghei malaria mouse model. 341
Because antifolate drugs such as pyrimethamine and proguanil have lost their efficacy due to 342
resistance, it is crucial for any new molecule targeting this enzyme to show activity against all 343
pyrimethamine-resistant DHFR mutant forms. In an earlier study, we assessed the in vitro 344
activity of WR99210 against the same clinical isolates tested in this publication (mean IC50 of 345
about 0.72 nM (19)). Thus when compared to QN254, WR99210 is about 10 times more potent 346
against parasite despite having a slightly lower binding affinity to PfDHFR than QN254. It 347
remains to be determined whether better diffusion across membranes of parasitized red blood 348
cells and/or an additional off-target effect could explain WR99210 higher cellular potency. 349
Using a functional/biochemical assay and crystallographic structural data, we provide further 350
evidence that QN254 binds to and inhibits Plasmodium WT and QM DHFR enzymes 351
efficiently. The resolution of the three-dimensional structure of wild-type (WT) and QM 352
PfDHFR – with either PM or WR99210 bound to its active site – provided structural insights 353
into DHFR pyrimethamine resistance mechanisms as well as some understanding of the 354
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
19
structural features allowing WR99210 to retain affinity for QM PfDHFR (55, 56). Our 355
crystallographic data, describing the structure of a ternary complex of QN254 bound to QM 356
PfDHFR, show that QN254 binds to its target in a manner similar to WR99210. Previous X-ray 357
crystallographic studies (5) with a compound closely related to QN254 and the Pneumocystis 358
carinii DHFR enzyme suggested that Phe 69 (P. carinii numbering or Phe 116 in Pf 359
numbering) was engaged in close hydrophobic contact with the 5′ methoxy group on the 360
phenyl ring of the lipophilic tail. The authors also speculated that this interaction may 361
contribute to the relative selectivity toward P. carinii (and possibly P. falciparum) as the 362
conserved phenylalanine was not present in the mammalian DHFR enzymes. Careful analysis 363
of our co-crystal QN254/PfDHFR structure failed to reveal such an interaction and in fact we 364
could only identify weak van der Waals interactions between the lipophilic tail and the 365
hydrophobic cavity present in the active site of QM PfDHFR. The structure activity 366
relationship (SAR) data for the class of quinazoline compounds are not very clear and are 367
currently limited to what is described in previous publications (8-11, 38, 39). 368
Consistent with our structural data demonstrating the absence of specific interactions between 369
the phenyl ring tail and PfDHFR, the available SAR data show that a large number of 370
modifications of the lipophilic tails are tolerated. With respect to selectivity, our data do not 371
provide any obvious structural or molecular explanation to rationalize the relative selectivity of 372
QN254 for P. falciparum versus the human enzyme. 373
For more than 30 years, it has been known that QN derivatives inhibit the growth and 374
proliferation of eukaryotic parasites such as Toxoplasma, Pneumocystis, Trypanosoma and 375
Plasmodium (8, 10, 11, 23, 36, 38, 45, 46). In fact, an extensive series of quinazolines have 376
previously proved to be efficacious in rodent and simian malaria models (41-44). The most 377
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
20
advanced candidate of this series, WR158122, entered clinical development as a candidate 378
antimalarial drug but was later abandoned because of its relatively poor oral efficacy in humans 379
(43). 380
Following oral administration QN254 is well absorbed, displays a long half-life and an 381
excellent oral bioavailability in rodents as well as in larger species such as dog (data not 382
shown). Although PAMPA and Caco-2 in vitro permeability data were predictive of the good 383
absorption and bioavailability, our in vitro metabolic stability data predicted that QN254 would 384
be subject to rapid hepatic clearance in all preclinical species as well as in humans (data not 385
shown). Nonetheless, in vivo QN254 showed a low (rats and dogs) to moderate (mouse) 386
systemic clearance in all preclinical species. We cannot explain the discrepancy between in 387
vitro and in vivo hepatic clearance and further studies would be required to determine the main 388
clearance mechanism in vivo. 389
Consistent with our pharmacokinetics results, QN254 displays good efficacy in the P. berghei 390
mouse model through the oral route. However, in P. berghei infected mice, we have found 391
evidence of toxicity upon repeated daily dosing for 3 days at doses as low as 60 mg/kg and 392
indeed further rat toxicology studies showed that QN254 does not possess an adequate 393
therapeutic window. At this juncture we cannot unambiguously establish whether the observed 394
toxicity is due to on- or off-target effects; and as an example we indeed have evidence that 395
QN254 presents some inherent risk of cardiotoxicity because of its ability to bind and inhibit 396
the hERG channel in vitro (hERG IC50 = 1.4 µM). Nonetheless, the rat histopathological data 397
showing that a highly proliferative tissue such as the bone marrow is massively affected is 398
consistent with on-target toxicity and sustained inhibition of folates metabolism through DHFR 399
inhibition (50, 54). Thus, despite its relative selectivity toward plasmodium DHFR enzyme in 400
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
21
vitro, QN254 appears to potently inhibit mammalian (rat) DHFR in vivo. Based on the 401
exposure reached at the efficacious dose and the exposure reached in rats upon dosing at 50 402
mg/kg in the toxicology study, we estimated that the therapeutic window is less than two. A 403
narrow therapeutic window is indeed a hallmark of almost all antifolates and has historically 404
been a significant hurdle to the development of new therapies based on DHFR inhibition. PM, 405
for example, displays an acute LD50 of 130 mg/kg in mice (50). Mitigating this toxicity risk is 406
the fact that antifolates such as PM are commonly used in combination at low doses (<5 mg/kg 407
in humans). Because of the severe toxicity observed in rats, the Novartis Institute for Tropical 408
Diseases is no longer pursuing the development of QN254. Nevertheless, further research into 409
the feasibility of strategies—e.g. pro-drug approach, supplementation with folate derivatives 410
such as 5-methyl-THF (27), or combination with antimalarials showing synergistic effects with 411
DHFR inhibitors—aiming to increase the therapeutic window of QN254 may be warranted. 412
413
6 Acknowledgments 414
We thank the director of the Kenya Medical Research Institute for permission to publish these 415
data. SK is an international research scholar of Howard Hughes Medical Institute (HHMI), 416
USA. KEMRI received support from the EU Commission under Framework 6 as part of the 417
AntiMal Integrated Project 018834, the European & Developing Countries Clinical Trials 418
Partnership (EDCTP) and the Wellcome Trust WT077092. BIOTEC was supported by grants 419
from Thailand-TDR (T-2) and the Medicines for Malaria Venture. NITD and STI receive 420
funding from a joint grant of the Medicines for Malaria Venture and the Wellcome Trust 421
(WT078285). 422
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
22
423
7 References 424
1. Brunger, A. T., P. D. Adams, G. M. Clore, W. L. DeLano, P. Gros, R. W. Grosse-Kunstleve, J. S. 425
Jiang, J. Kuszewski, M. Nilges, N. S. Pannu, R. J. Read, L. M. Rice, T. Simonson, and G. L. 426
Warren. 1998. Crystallography & NMR system: A new software suite for macromolecular structure 427
determination. Acta Crystallogr. D 54 ( Pt 5):905-921. 428
2. Canfield, C. J., W. K. Milhous, A. L. Ager, R. N. Rossan, T. R. Sweeney, N. J. Lewis, and D. P. 429
Jacobus. 1993. PS-15: a potent, orally active antimalarial from a new class of folic acid antagonists. Am. 430
J. Trop. Med. Hyg. 49:121-126. 431
3. Childs, G. E., and C. Lambros. 1986. Analogues of N-benzyloxydihydrotriazines: in vitro antimalarial 432
activity against Plasmodium falciparum. Ann. Trop. Med. Parasitol. 80:177-181. 433
4. Chitnumsub, P., J. Yuvaniyama, J. Vanichtanankul, S. Kamchonwongpaisan, M. D. Walkinshaw, 434
and Y. Yuthavong. 2004. Characterization, crystallization and preliminary X-ray analysis of 435
bifunctional dihydrofolate reductase-thymidylate synthase from Plasmodium falciparum. Acta 436
Crystallogr. D Biol. Crystallogr. 60:780-783. 437
5. Cody, V., N. Galitsky, J. R. Luft, W. Pangborn, S. F. Queener, and A. Gangjee. 2002. Analysis of 438
quinazoline and pyrido[2,3-d]pyrimidine N9-C10 reversed-bridge antifolates in complex with NADP+ 439
and Pneumocystis carinii dihydrofolate reductase. Acta Crystallogr D Biol Crystallogr 58:1393-1399. 440
6. Delano W L. 2002. The PyMOL Molecular Graphics System. The PyMOL Molecular Graphics 441
System.:Delano Scientific, Palo Alto, CA. 442
7. Dondorp, A. M., F. Nosten, P. Yi, D. Das, A. P. Phyo, J. Tarning, K. M. Lwin, F. Ariey, W. 443
Hanpithakpong, S. J. Lee, P. Ringwald, K. Silamut, M. Imwong, K. Chotivanich, P. Lim, T. 444
Herdman, S. S. An, S. Yeung, P. Singhasivanon, N. P. Day, N. Lindegardh, D. Socheat, and N. J. 445
White. 2009. Artemisinin resistance in Plasmodium falciparum malaria. N. Engl. J. Med. 361:455-467. 446
8. Elslager, E. F., O. D. Bird, J. Clarke, S. C. Perricone, and D. F. Worth. 1972. Folate antagonists. 9. 447
2,4-Diamino-6-((aralkyl)alkylamino)quinazolines, a potent class of antimetabolites with prodigious 448
antimalarial effects. J. Med. Chem. 15:1138-1146. 449
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
23
9. Elslager, E. F., J. Clarke, L. M. Werbel, D. F. Worth, and J. Davoll. 1972. Folate antagonists. 3. 2,4-450
Diamino-6-(heterocyclic)quinazolines, a novel class of antimetabolites with potent antimalarial and 451
antibacterial activity. J. Med. Chem. 15:827-836. 452
10. Elslager, E. F., M. P. Hutt, P. Jacob, J. Johnson, B. Temporelli, L. M. Werbel, D. F. Worth, and L. 453
Rane. 1979. Folate antagonists. 15. 2,3-Diamino-6-(2-naphthylsulfonyl)quinazoline and related 2,4-454
diamino-6-[(phenyl and naphthyl)sulfinyl and sulfonyl]quinazolines, a potent new class of 455
antimetabolites with phenomenal antimalarial activity. J. Med. Chem. 22:1247-1257. 456
11. Elslager, E. F., P. Jacob, J. Johnson, L. M. Werbel, D. F. Worth, and L. Rane. 1978. Folate 457
antagonists. 13. 2,4-Diamino-6-](alpha,alpha,alpha-trifluoro-m-tolyl)thio]quinazoline and related 2,4-458
diamino-6-[(phenyl- and naphthyl)thio]quinazolines, a unique class of antimetabolites with extraordinary 459
antimalarial and antibacterial effects. J. Med. Chem. 21:1059-1070. 460
12. Fidock, D. A., P. J. Rosenthal, S. L. Croft, R. Brun, and S. Nwaka. 2004. Antimalarial drug 461
discovery: efficacy models for compound screening. Nat. Rev. Drug Discov. 3:509-520. 462
13. Gangjee, A., S. Kurup, and O. Namjoshi. 2007. Dihydrofolate reductase as a target for chemotherapy 463
in parasites. Curr. Pharm. Des. 13:609-639. 464
14. Gregson, A., and C. V. Plowe. 2005. Mechanisms of resistance of malaria parasites to antifolates. 465
Pharmacol. Rev. 57:117-145. 466
15. Jackman, A. L., F. T. Boyle, and K. R. Harrap. 1996. Tomudex (ZD1694): from concept to care, a 467
programme in rational drug discovery. Invest. New Drugs 14:305-316. 468
16. Jones, T. A., J. Y. Zou, S. W. Cowan, and M. Kjeldgaard. 1991. Improved methods for building 469
protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 470
47:110-119. 471
17. Kamchonwongpaisan, S., R. Quarrell, N. Charoensetakul, R. Ponsinet, T. Vilaivan, J. 472
Vanichtanankul, B. Tarnchompoo, W. Sirawaraporn, G. Lowe, and Y. Yuthavong. 2004. Inhibitors 473
of multiple mutants of Plasmodium falciparum dihydrofolate reductase and their antimalarial activities. J 474
Med. Chem. 47:673-680. 475
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
24
18. Kamchonwongpaisan, S., J. Vanichtanankul, B. Tarnchompoo, J. Yuvaniyama, S. Taweechai, and 476
Y. Yuthavong. 2005. Stoichiometric selection of tight-binding inhibitors by wild-type and mutant forms 477
of malarial (Plasmodium falciparum) dihydrofolate reductase. Anal. Chem. 77:1222-1227. 478
19. Kiara, S. M., J. Okombo, V. Masseno, L. Mwai, I. Ochola, S. Borrmann, and A. Nzila. 2009. In vitro 479
activity of antifolate and polymorphism in dihydrofolate reductase of Plasmodium falciparum isolates 480
from Kenyan coast: Emergence of parasites with Ile-164-Leu mutation. Antimicrob. Agents Chemother. 481
20. Kinyanjui, S. M., E. K. Mberu, P. A. Winstanley, D. P. Jacobus, and W. M. Watkins. 1999. The 482
antimalarial triazine WR99210 and the prodrug PS-15: folate reversal of in vitro activity against 483
Plasmodium falciparum and a non-antifolate mode of action of the prodrug. Am. J. Trop. Med. Hyg. 484
60:943-947. 485
21. Kleywegt G J. 1997. Validation of protein models from CA coordinates alone. J. Mol. Biol. 273:371-486
376. 487
22. Knight, D. J., P. Mamalis, and W. Peters. 1982. The antimalarial activity of N-benzyl-488
oxydihydrotriazines. III. The activity of 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(2,4,5,-489
trichloropropyloxy)-1,3,5-t riazine hydrobromide (BRL 51084) and hydrochloride (BRL 6231). Ann 490
Trop. Med. Parasitol. 76:1-7. 491
23. Kovacs, J. A., C. J. Allegra, B. A. Chabner, J. C. Swan, J. Drake, M. Lunde, J. E. Parrillo, and H. 492
Masur. 1987. Potent effect of trimetrexate, a lipid-soluble antifolate, on Toxoplasma gondii. J. Infect. 493
Dis. 155:1027-1032. 494
24. Laskowski R A, MacArthur M W, Moss D S, and Thronton J M. 1993. PROCHECK: a program to 495
check the stereochemical quality of protein structures. . J. Appl. Cryst. 26:283-291. 496
25. Morris A L, MacArthur M W, Hutchinson E G, and Thronton J M. 1992. Stereochemical quality of 497
protein structure coordinates. . Proteins. 12:345-364. 498
26. Murshudov G N, Vagin A A, and Dodson E J. 1997. Refinement of macromolecular structures by the 499
maximum-likelihood method. Acta Crystallogr. D 53:240-255. 500
27. Nduati, E., A. Diriye, S. Ommeh, L. Mwai, S. Kiara, V. Masseno, G. Kokwaro, and A. Nzila. 2008. 501
Effect of folate derivatives on the activity of antifolate drugs used against malaria and cancer. Parasitol. 502
Res. 102:1227-1234. 503
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
25
28. Noedl, H., Y. Se, K. Schaecher, B. L. Smith, D. Socheat, and M. M. Fukuda. 2008. Evidence of 504
artemisinin-resistant malaria in western Cambodia. N. Engl. J. Med. 359:2619-2620. 505
29. Nzila, A. 2006. Inhibitors of de novo folate enzymes in Plasmodium falciparum. Drug Discov. Today 506
11:939-944. 507
30. Nzila, A. 2006. The past, present and future of antifolates in the treatment of Plasmodium falciparum 508
infection. J Antimicrob. Chemother. 57:1043-1054. 509
31. Nzila, A. M., E. K. Mberu, J. Sulo, H. Dayo, P. A. Winstanley, C. H. Sibley, and W. M. Watkins. 510
2000. Towards an understanding of the mechanism of pyrimethamine-sulfadoxine resistance in 511
Plasmodium falciparum: genotyping of dihydrofolate reductase and dihydropteroate synthase of Kenyan 512
parasites. Antimicrob. Agents Chemother. 44:991-996. 513
32. O'Dwyer, P. J., R. J. DeLap, S. A. King, A. J. Grillo-Lopez, D. F. Hoth, and B. Leyland-Jones. 514
1987. Trimetrexate: clinical development of a nonclassical antifolate. NCI Monogr:105-109. 515
33. O'Dwyer, P. J., D. D. Shoemaker, J. Plowman, J. Cradock, A. Grillo-Lopez, and B. Leyland-Jones. 516
1985. Trimetrexate: a new antifol entering clinical trials. Invest. New Drugs 3:71-75. 517
34. Ommeh, S., E. Nduati, E. Mberu, G. Kokwaro, K. Marsh, A. Rosowsky, and A. Nzila. 2004. In vitro 518
activities of 2,4-diaminoquinazoline and 2,4-diaminopteridine derivatives against Plasmodium 519
falciparum. Antimicrob. Agents Chemother. 48:3711-3714. 520
35. Otwinowski, Z. M., and W. Minor. 1979. Processing of X-ray diffraction data collected in oscillation 521
mode. Method Enzymol. 276:307-326. 522
36. Piper, J. R., C. A. Johnson, C. A. Krauth, R. L. Carter, C. A. Hosmer, S. F. Queener, S. E. Borotz, 523
and E. R. Pfefferkorn. 1996. Lipophilic antifolates as agents against opportunistic infections. 1. Agents 524
superior to trimetrexate and piritrexim against Toxoplasma gondii and Pneumocystis carinii in in vitro 525
evaluations. J. Med. Chem. 39:1271-1280. 526
37. Ridley, R. G., H. Matile, C. Jaquet, A. Dorn, W. Hofheinz, W. Leupin, R. Masciadri, F. P. Theil, 527
W. F. Richter, M. A. Girometta, A. Guenzi, H. Urwyler, E. Gocke, J. M. Potthast, M. Csato, A. 528
Thomas, and W. Peters. 1997. Antimalarial activity of the bisquinoline trans-N1,N2-bis (7-529
chloroquinolin-4-yl)cyclohexane-1,2-diamine: comparison of two stereoisomers and detailed evaluation 530
of the S,S enantiomer, Ro 47-7737. Antimicrob. Agents Chemother. 41:677-686. 531
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
26
38. Rosowsky, A., C. E. Mota, J. E. Wright, J. H. Freisheim, J. J. Heusner, J. J. McCormack, and S. F. 532
Queener. 1993. 2,4-Diaminothieno[2,3-d]pyrimidine analogues of trimetrexate and piritrexim as 533
potential inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase. J. Med. 534
Chem. 36:3103-3112. 535
39. Rosowsky, A., C. E. Mota, J. E. Wright, and S. F. Queener. 1994. 2,4-Diamino-5-chloroquinazoline 536
analogues of trimetrexate and piritrexim: synthesis and antifolate activity. J. Med. Chem. 37:4522-4528. 537
40. Sasi, P., A. Abdulrahaman, L. Mwai, S. Muriithi, J. Straimer, E. Schieck, A. Rippert, M. 538
Bashraheil, A. Salim, J. Peshu, K. Awuondo, B. Lowe, M. Pirmohamed, P. Winstanley, S. Ward, A. 539
Nzila, and S. Borrmann. 2009. In vivo and in vitro efficacy of amodiaquine against Plasmodium 540
falciparum in an area of continued use of 4-aminoquinolines in East Africa. J. Infect. Dis. 199:1575-541
1582. 542
41. Schmidt, L. H. 1978. Plasmodium falciparum and Plasmodium vivax infections in the owl monkey 543
(Aotus trivirgatus). III. Methods employed in the search for new blood schizonticidal drugs. Am. J. Trop. 544
Med. Hyg. 27:718-737. 545
42. Schmidt, L. H. 1979. Studies on the 2,4-diamino-6-substituted quinazolines. II. Activities of selected 546
derivatives against infections with various drug-susceptible and drug-resistant strains of Plasmodium 547
falciparum and Plasmodium vivax in owl monkeys. Am. J. Trop. Med. Hyg. 28:793-807. 548
43. Schmidt, L. H. 1979. Studies on the 2,4-diamino-6-substituted quinazolines. III. The capacity of 549
sulfadiazine to enhance the activities of WR-158,122 and WR-159,412 against infections with various 550
drug-susceptible and drug-resistant strains of Plasmodium falciparum and Plasmodium vivax in owl 551
monkeys. Am. J. Trop. Med. Hyg. 28:808-818. 552
44. Schmidt, L. H., and R. N. Rossan. 1979. Studies on the 2,4-diamino-6 substituted quinazolines. I. 553
Antimalarial activities of 2,4-diamino-6-[(3,4-dichlorobenzyl)-nitrosoamino]-quinazoline (CI-679) as 554
exhibited in rhesus monkeys infected with the Ro or Ro/PM strains of Plasmodium cynomolgi. Am. J. 555
Trop. Med. Hyg. 28:781-792. 556
45. Senkovich, O., V. Bhatia, N. Garg, and D. Chattopadhyay. 2005. Lipophilic antifolate trimetrexate is 557
a potent inhibitor of Trypanosoma cruzi: prospect for chemotherapy of Chagas' disease. Antimicrob. 558
Agents Chemother. 49:3234-3238. 559
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
27
46. Senkovich, O., B. Pal, N. Schormann, and D. Chattopadhyay. 2003. Trypanosoma cruzi genome 560
encodes a pteridine reductase 2 protein. Mol. Biochem .Parasitol. 127:89-92. 561
47. Sibley, C. H., J. E. Hyde, P. F. Sims, C. V. Plowe, J. G. Kublin, E. K. Mberu, A. F. Cowman, P. A. 562
Winstanley, W. M. Watkins, and A. M. Nzila. 2001. Pyrimethamine-sulfadoxine resistance in 563
Plasmodium falciparum: what next? Trends Parasitol. 17:582-588. 564
48. Sirawaraporn, W., R. Sirawaraporn, S. Yongkiettrakul, A. Anuwatwora, G. Rastelli, S. 565
Kamchonwongpaisan, and Y. Yuthavong. 2002. Mutational analysis of Plasmodium falciparum 566
dihydrofolate reductase: the role of aspartate 54 and phenylalanine 223 on catalytic activity and antifolate 567
binding. Mol. Biochem. Parasitol. 121:185-193. 568
49. Sixsmith, D. G., W. M. Watkins, J. D. Chulay, and H. C. Spencer. 1984. In vitro antimalarial activity 569
of tetrahydrofolate dehydrogenase inhibitors. Am. J. Trop. Med. Hyg. 33:772-776. 570
50. Stephan-Guldner, M. 1993. Preclinical toxicology and safety pharmacology of brodimoprim in 571
comparison to trimethoprim and analogs. J. Chemother. 5:400-410. 572
51. Tarnchompoo, B., C. Sirichaiwat, W. Phupong, C. Intaraudom, W. Sirawaraporn, S. 573
Kamchonwongpaisan, J. Vanichtanankul, Y. Thebtaranonth, and Y. Yuthavong. 2002. 574
Development of 2,4-diaminopyrimidines as antimalarials based on inhibition of the S108N and 575
C59R+S108N mutants of dihydrofolate reductase from pyrimethamine-resistant Plasmodium falciparum. 576
J. Med. Chem. 45:1244-1252. 577
52. Vennerstrom, J. L., S. Arbe-Barnes, R. Brun, S. A. Charman, F. C. Chiu, J. Chollet, Y. Dong, A. 578
Dorn, D. Hunziker, H. Matile, K. McIntosh, M. Padmanilayam, J. Santo Tomas, C. Scheurer, B. 579
Scorneaux, Y. Tang, H. Urwyler, S. Wittlin, and W. N. Charman. 2004. Identification of an 580
antimalarial synthetic trioxolane drug development candidate. Nature 430:900-904. 581
53. White, N. J. 2008. Qinghaosu (artemisinin): the price of success. Science 320:330-334. 582
54. Winstanley, P. A., E. K. Mberu, I. S. Szwandt, A. M. Breckenridge, and W. M. Watkins. 1995. In 583
vitro activities of novel antifolate drug combinations against Plasmodium falciparum and human 584
granulocyte CFUs. Antimicrob. Agents Chemother. 39:948-952. 585
55. Yuthavong, Y., J. Yuvaniyama, P. Chitnumsub, J. Vanichtanankul, S. Chusacultanachai, B. 586
Tarnchompoo, T. Vilaivan, and S. Kamchonwongpaisan. 2005. Malarial (Plasmodium falciparum) 587
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
28
dihydrofolate reductase-thymidylate synthase: structural basis for antifolate resistance and development 588
of effective inhibitors. Parasitol. 130:249-259. 589
56. Yuvaniyama, J., P. Chitnumsub, S. Kamchonwongpaisan, J. Vanichtanankul, W. Sirawaraporn, P. 590
Taylor, M. D. Walkinshaw, and Y. Yuthavong. 2003. Insights into antifolate resistance from malarial 591
DHFR-TS structures. Nat. Struct. Biol. 10:357-365. 592
593
594
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
29
Figure legends 595
596
Figure 1. Co-crystal structure of dihydrofolate reductase quadruple mutant (mutations at 597
codons 108, 51, 59, 164) showing H-bond interactions with QN254 (A) and WR99210 (B). 598
Both complexes are superposed in (C). 599
600
601
Figure 2. Pharmacokinetic profile of QN254 upon intravenous and oral administration – 602
QN254 was administered orally at 25 mg/kg (filled diamonds) and intravenously at 5 mg/kg 603
(open squares) to mice (A) and rats (B). Plasma concentrations were measured over time and 604
are indicated in ng/ml ± standard deviations (n=3). Note that the timescale is different in A and 605
B. Drug levels for the last time points in profile A were detectable but below the lower limit of 606
quantification (12 ng/ml). 607
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
30
608
Table legends 609
610
Table 1. Results of competitive binding kinetics experiments on DHFR enzymes of P. 611
falciparum wild-type (WT-PfDHFR) and quadruple mutant (QM-PfDHFR) [S108N, N51I, 612
C59R, and I164L], and human (hDHFR) against QN254, WR99210, pyrimethamine (PM) and 613
cycloguanil (CLG). Ki, binding constant (nM) 614
615
616
Table 2. Relationship between pyrimethamine (PM) and QN254 median inhibitory 617
concentrations that kill 50% of parasitemia (IC50) and dihydrofolate reductase (dhfr) genotypes 618
of clinical isolates. No wild-type dhfr isolate was identified; n, number of isolates. Double 619
mutants are S108N and N51I or S108N and C59R; triple mutants are S108, N51I and C59R; 620
and quadruple mutants are S108N, N51, C59R and Ile164Leu 621
622
623
Table 3. QN254 antimalarial activity in the Plasmodium. berghei murine malaria model. 624
Results represent % inhibition of parasitemia compared to untreated controls. QN254 was 625
prepared in 0.5% carboxymethyl cellulose; Survival of control animals: 5.6–6.2 days; * Mice 626
died around day 10, and were parasite-free; # Solution formulation with 10% ethanol, 30% 627
PEG400 (polyethylene glycol 400) and 60% of a 10% Vitamin ETPGS solution. N/A not 628
available; n≥10 mice except for the three times treatment with QN254 629
630
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
Table 1:
Ki (nM) Ki ratio
WT-PfDHFR
QM-
PfDHFR
hDHFR hDHFR/WT-
pfDHFR QM-
PfDHFR/WT-
PfDHFR
CLG 1.51 ± 0.1 454 ± 38 55.6 ± 7.8 37 300
PM 0.59 ± 0.05 385 ± 163 30.8 ± 1.4 52 652
WR99210 0.5 ± 0.1 1.9 ± 0.8 7.7 ± 0.4 15 1.8
QN254 0.39 ± 0.05 0.58 ± 0.06 10.2 ± 0.6 26 1.5
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
(A)
(B)
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
(C)
Figure 1:
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
Figure 2.
1
10
100
1000
10000
0 5 10 15 20 25
Time (h)
Mean C
oncentr
ation
(ng
/ml)
1
10
100
1000
10000
0 5 10 15 20 25
Time (h)
Mean C
oncentr
ation
(ng
/ml)
A
B
1
10
100
1000
10000
0 10 20 30 40 50
Time (h)
Mean C
oncentr
ation
(ng
/ml)
1
10
100
1000
10000
0 10 20 30 40 50
Time (h)
Mean C
oncentr
ation
(ng
/ml)
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
Table 2:
.
*Data pertaining to PM were already published elsewhere (38).
**None of the tested differences between double and triple mutants were significant at p=0.05 (Kruskall–
Wallis test).
DHFR genotype IC50 (n)
nM
PM* QN254
Double mutant** 371 (8) 4.48 (6)
Triple mutant** 778.28 (24) 11.66 (20)
Quadruple mutant 3690.79 (1) 7.61 (1)
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
Table 3:
Dosing regimen Parasitemia
reduction (%)
Average mouse
survival (days)
Cure rate
(%)
Toxicity rate
(%)
QN254
1 x 10 mg/kg 59 6.0 0 0
1 x 15 mg/kg 73 6.0 0 0
1 x 30 mg/kg >99.99 8.1 0 0
1 x 60 mg/kg >99.99 12.0 0 0
1 x 100 mg/kg >99.99 23.4 50 0
3 x 30 mg/kg >99.99 28.4 80 0
3 x 60 mg/kg >99.99 N/A 60 40*
3 x 100 mg/kg >99.99 N/A 20 80*
Artesunate#
1 x 100 mg/kg 98 7.0 0 0
3 x 30 mg/kg 98.7 12.2 0 0
Chloroquine#
1 x 100 mg/kg 99.6 12.5 0 0
3 x 30 mg/kg 99.8 14.3 0 0
Mefloquine#
1 x 100 mg/kg 89 25.5 0 0
3 x 30 mg/kg 98.8 22.1 0 0
Results represent % inhibition of parasitemia compared to untreated controls; QN254 was
prepared in 0.5% carboxymethyl cellulose; Survival of control animals: 5.6–6.2 days; * Mice
died around day 10, and were parasite-free; #や Solution formulation with 10% ethanol, 30%
PEG400 (polyethylene glycol 400) and 60% of a 10% Vitamin ETPGS solution. N/A not
available; nœ10 mice except for the three times treatment with QN254 because of ethical reasons
due to the observed toxicity
on June 9, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
