1
Atovaquone and ELQ-300 combination therapy: 1
A novel dual-site cytochrome bc1 inhibition strategy for malaria 2
3
Allison M. Stickles1#, Martin J. Smilkstein1, Joanne M. Morrisey3, Yuexin Li2, Isaac P. Forquer2, 4
Jane X. Kelly2, Sovitj Pou2, Rolf W. Winter2, Aaron Nilsen2, Akhil B. Vaidya3, & Michael K. 5
Riscoe1,2# 6
7
1Departments of Physiology and Pharmacology, Molecular Microbiology and Immunology, and 8
Emergency Medicine, Oregon Health & Science University, Portland, OR, USA. 2VA Medical 9
Center, Portland, OR, USA. 3Center for Molecular Parasitology, Department of Microbiology 10
and Immunology, Drexel University College of Medicine, Philadelphia, PA, USA. 11
12
Running Head: ATV and ELQ-300 combination therapy 13
14
# Address correspondence to Allison M. Stickles: [email protected] or Michael K. Riscoe: 15
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AAC Accepted Manuscript Posted Online 6 June 2016Antimicrob. Agents Chemother. doi:10.1128/AAC.00791-16Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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Abstract 24
Antimalarial combination therapies play a crucial role in preventing the emergence of drug 25
resistant Plasmodium parasites. Although artemisinin-based combination therapies (ACTs) 26
comprise the majority of these formulations, inhibitors of the mitochondrial cytochrome bc1 27
complex (cyt bc1) are among the few compounds that are effective for both acute antimalarial 28
treatment and prophylaxis. There are two known sites for inhibition within cyt bc1: atovaquone 29
(ATV) blocks the quinone oxidase (Qo) site of cyt bc1, while some members of the endochin-like 30
quinolone (ELQ) family, including preclinical candidate ELQ-300, inhibit the quinol reductase 31
(Qi) site, and retain full potency against ATV-resistant P. falciparum strains with Qo site 32
mutations. Here, we provide the first in vivo comparison of ATV, ELQ-300, and ATV:ELQ-300 33
combination therapy, using P. yoelii murine models of malaria. In our monotherapy assessments, 34
we found that ATV functioned as a single dose curative compound in suppressive tests while 35
ELQ-300 demonstrated a unique cumulative dosing effect that successfully blocked 36
recrudescence even in a high parasitemia, acute infection model. ATV:ELQ-300 therapy was 37
highly synergistic, and the combination was curative with a single combined dose of 1 mg/kg. As 38
compared to the ATV:proguanil formulation used in Malarone, ATV:ELQ-300 was more 39
efficacious in multi-day, acute infection models and was equally effective at blocking the 40
emergence of ATV resistant parasites. Ultimately, our data suggest that dual-site inhibition of cyt 41
bc1 is a valuable strategy for antimalarial combination therapy, and that Qi site inhibitors such as 42
ELQ-300 represent valuable partner drugs for the clinically successful Qo site inhibitor, ATV. 43
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Introduction 47
Malaria is a devastating tropical disease, which disproportionately affects children and pregnant 48
women and is responsible for more than 400,000 deaths each year (1). Although many 49
antimalarial drugs exist, treatment is complicated by the rapid emergence of drug resistance 50
among the Plasmodium parasites that cause disease. Mutations in PfCRT heralded resistance to 51
chloroquine in the late 1950s, and since that time, resistance has developed against compounds in 52
every major antimalarial class (2, 3). Even artemisinin-based combination therapies (ACTs), 53
which are the current mainstay of treatment in endemic regions, are slowly losing activity in 54
Southeast Asia (4). 55
56
Although the parasitology community continues to develop novel antimalarial compounds, 57
combinations of existing therapies have also been embraced as a way to prevent or overcome 58
drug resistance. The majority of drugs in current Phase III clinical trials are ACTs, which 59
typically partner a fast-acting artemisinin derivative with a long-duration antimalarial such as 60
piperaquine or mefloquine (5). In contrast, many antibacterial and anti-cancer combination 61
therapies prioritize the use of synergistic compounds targeting multiple steps of the same 62
biochemical pathway in an effort to minimize drug resistance (6, 7). In malaria, a promising 63
target for such an approach is the cytochrome bc1 complex (cyt bc1), which has recently been 64
identified as a target for single-dose blood and vector-stage antimalarial therapy, and is capable 65
of initiating rapid parasite clearance in vivo (8). 66
67
Cyt bc1 is the known site of action of several potent inhibitors of P. falciparum, including 68
atovaquone (ATV) (9) and the endochin-like quinolones (ELQs) (10). Biologically, cyt bc1 plays 69
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a vital role in de-novo pyrimidine biosynthesis for P. falciparum by facilitating the activity of 70
type II dihydroorotate dehydrogenase (DHODH) (11). Because malaria parasites lack a 71
functional pyrimidine salvage pathway, inhibition of cyt bc1 (and by extension, DHODH) is cidal 72
and provides both treatment and prophylactic protection against malaria (12). Currently, ATV is 73
the only cyt bc1 inhibitor in clinical use, and is a key component of the antimalarial combination 74
therapy, Malarone (13). 75
76
Although cyt bc1 can be effectively inhibited at either its quinol oxidase (Qo) or quinone 77
reductase (Qi) site, ATV is a selective Qo site inhibitor (14), and ATV-resistance is associated 78
with point mutations at this site, including the clinically-relevant Y268S substitution that reduces 79
sensitivity to ATV by more than 1000-fold (15, 16). Our group has recently shown that current 80
preclinical candidate ELQ-300 potently inhibits the Qi site of cyt bc1, rather than the Qo site 81
exploited by ATV (17). Because of this site specificity, ELQ-300 is fully active against ATV-82
resistant P. falciparum parasites containing Y268S mutations (18). Similarly, ELQ-300 resistant 83
P. falciparum parasites are fully sensitive to ATV in vitro (17). Because all known clinical cases 84
of ATV resistance are attributable to point mutations within the cytochrome b gene, this suggests 85
that ATV and ELQ-300 would be mutually protective against the generation of drug resistance 86
and would therefore represent an optimal choice for antimalarial combination therapy. 87
88
ATV:ELQ-300 combination therapy is also positioned to overcome several well-established 89
shortcomings of the ATV:proguanil formulation utilized in Malarone. In circulation, proguanil is 90
rapidly metabolized to cycloguanil, while ATV has a significantly longer half-life (19). Although 91
cycloguanil is an effective dihydrofolate reducase (DHFR) inhibitor, there have been reports of 92
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Malarone resistance involving simultaneous mutations in DHFR and cytochrome b (20, 21), and 93
only unmetabolized proguanil works synergistically with ATV to collapse the mitochondrial 94
membrane potential (11, 22, 23). Consequently and more concerningly, the Y268S mutation 95
associated with high-grade ATV resistance also eliminates ATV:proguanil synergy at the level of 96
the mitochondria (24). Therefore, despite the clinical success of Malarone as a prophylactic 97
agent, there are significant concerns that resistance would compromise its use in acute infections 98
or mass drug administration efforts (25). 99
100
In contrast, ELQ-300 and ATV have similar predicated half-lives in humans (~70 hours) (18), 101
eliminating the potential for ATV monotherapy and resultant ATVR mutations. Because both 102
ATV and ELQ-300 act on a single protein, the development of simultaneous Qo and Qi site 103
mutations would be exceedingly rare, and the fact that the mitochondrial genome is carried in 104
only female gametocytes would prevent any dual-site resistance secondary to genetic 105
recombination at the sexual stage. Even if rare ATVR/ELQ-300R mutants were to arise, recent 106
work suggests that these parasites would likely be incapable of transmission by mosquitoes: the 107
McFadden group has recently shown that multiple ATVR mutations (including Y268C and 108
Y268N) are associated with arrested oocyst development within the mosquito mid-gut, and a 109
failure to produce infectious sporozoites necessary for transmission to mammalian hosts (26). 110
Ultimately, we believe that ATV and ELQ-300 could be ideal partner drugs with a low 111
propensity for drug resistance, and the remarkable potency characteristic of cyt bc1 inhibitors. In 112
this manuscript, we characterize the single-dose and multi-dose efficacy of ATV, ELQ-300, and 113
combinations of ATV:ELQ-300 and ATV:proguanil in P. yoelii murine models. In addition, the 114
propensity for the development of drug resistance in the various treatment arms is described. 115
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116
Materials and Methods 117
In Vitro Synergy of ATV and ELQ-300 118
In vitro evaluation of ELQ-300 and ATV was assessed by isobolar analysis using a fixed-ratio 119
strategy (27). In each case, IC50 data was generated using a published SYBR Green I 120
fluorescence-based method (28) and FICs were then calculated using the following formula: FIC 121
(A) = IC50 of drug A in combination / IC50 of drug A alone; FIC (B) = IC50 of drug B in 122
combination / IC50 of drug B alone; FIC index = FIC (A) + FIC (B). Experiments were 123
performed in duplicate and isobolograms were constructed by plotting mean FIC + SEM for each 124
combination of ATV and ELQ-300. A straight diagonal line (FIC index = 1) indicates an additive 125
effect between the two drugs. A concave curve (FIC index < 1) indicates synergy of the 126
combination, and a convex curve (FIC index > 1) indicates antagonism. 127
128
Compounds and Formulation 129
ELQ-300 was synthesized and purified using previously published methods (10). ATV was 130
obtained from Sigma and recrystallized from DMF and methanol before use. All drug stocks 131
were prepared as 10 mg/kg stock solutions in PEG-400, and serially diluted such that the desired 132
concentration could be delivered in 100μL aliquots. For combination therapies, each drug was 133
stored separately, and animals received doses via sequential gavage. Reported doses represent 134
total drug present (e.g., a 20 mg/kg ATV:ELQ-300 dose would represent either 10 mg/kg ATV + 135
10 mg/kg ELQ-300 in the 1:1 formulation, or 15 mg/kg ATV + 5 mg/kg ELQ-300 in the 3:1 136
formulation). 137
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Peters Suppressive Test 139
Six-week old, female CF-1 mice were obtained from Charles River and infected with 2.5x105 140
blood-stage P. yoelii (Kenya strain, MR4 MRA-428) parasites via tail vein injection (N=4 141
animals/group). Drug stocks were administered to animals once daily for 1 or 4 days, beginning 142
24 hours post-infection. For ATV, ATV:ELQ-300, and ATV:proguanil groups, tested doses were 143
0.001, 0.01, 0.1, 1, and 10 mg/kg. Single-dose studies with ELQ-300 included an additional 20 144
mg/kg dosing group, and multi-dose ELQ-300 studies included additional doses at 0.003, 0.03, 145
and 0.3 mg/kg. In all groups, total dose was delivered via two 100μL boluses. After treatment, 146
daily blood samples were collected from the tail vein, beginning on post-infection day 5, and 147
parasitemia was determined microscopically using Giemsa stain and NIS-Elements cell-counting 148
software (Nikon, Melville, NY). Animals were treated in accordance with IACUC guidelines, 149
and were sacrificed when parasitemia exceeded 30%. ED50 was calculated as the dose that 150
effectively reduced day 5 parasitemia by 50% relative to controls. Animals were considered 151
cured if no parasitemia was detectable at post-infection day 30. 152
153
Acute Infection Model 154
Mice were infected as above, and blood was monitored daily via thin smear until parasitemia 155
reached 15-20%. At that time, drugs were administered via oral gavage as solutions in PEG-400, 156
and parasitemia was monitored daily via thin smear and plotted as a function of time post-157
treatment. Smears were collected daily for the first 60 days post-infection, and then 158
weekly/intermittently for at least 4 additional months. For animals that recrudesced post-159
treatment, resistance was assessed by re-treating animals with a second 20 mg/kg drug dose (at 160
10-25% parasitemia) and comparing parasite response to initial kinetics, lag time, and clearance 161
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time. Resistant parasites were collected via cardiac puncture, frozen in 30% glycerol, and the 162
cytochrome b gene was sequenced as below. 163
164
Cytochrome b Sequencing 165
DNA was isolated from parasites in mid to late trophozoite stage. Parasites were saponin lysed 166
with .02% saponin PBS. Pellets were resuspended to volume of 200μl with PBS/20mM EDTA 167
and DNA was isolated using the QIAamp DNA Blood mini kit (Qiagen) following the blood 168
protocol. PCR was performed using Herculase II Fusion enzyme (Agilent Technologies). Primer 169
1: 5’ CCAGACGCTTTAAATGGATG 3’. Primer2: 5’GTTTGCTTGGGAGCTGTAATC 3’. 170
PCR cleanup was performed using SV gel and PCR cleanup kit (Promega). Direct sequencing 171
by Genewiz. 172
173
In Vivo Assessment of ATVR and ELQ-300R Mutants 174
Mice (6-week, female, CF-1, Charles River) were infected with 2.5 × 105 P. yoelii-WT (Kenya 175
strain, MR4 MRA-428), P. yoelii-ATVRBY268C, or P. yoelii ELQ-300RI22L parasitized RBCs by 176
tail vein injection and 4-day Peters suppressive tests were conducted as above (N=4 177
animals/group). On post-infection Day 5, samples were collected from the tail vein and 178
parasitemia was determined by flow cytometry after blood sample incubation with SYBR Green 179
I, APC labeled anti-CD45, and dihydroethidium (29). Fifty percent (50%) effective dose (ED50) 180
values were defined as the dose required to reduce parasite burden by 50% relative to controls on 181
post-infection day 5. Giemsa-stained blood smears were used to determine the presence or 182
absence of parasites if flow cytometry indicated parasitemia less than 0.3%, and parasite-free 183
mice were maintained and monitored semi-weekly. Animals were considered cured if they 184
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remained parasite-free by microscopy until post-infection Day 30. The Portland VA Medical 185
Center Institutional Animal Care and Use Committee approved all protocols involving animals 186
used in this study. 187
188
Results 189
In Vitro Synergy of ATV and ELQ-300 190
In order to determine if ATV and ELQ-300 (Figure 1A) acted synergistically in vitro, we tested 191
these compounds against wild-type (D6) P. falciparum parasites, using a fixed-ratio, isobolar 192
strategy (27). As illustrated in Figure 1B, parasites were more sensitive to the combination of 193
ATV:ELQ-300 than to higher concentrations of either drug alone, with a mean FIC index of 194
0.65. 195
196
Peters Suppressive Test 197
We next used in vivo suppressive models to test the activity of ATV, ELQ-300, and ATV:ELQ-198
300 combinations in both 4-day and 1-day dosing tests (Table 1). For these studies, mice were 199
infected with P. yoelii parasites, and received the first oral drug dose 24 hours post-infection. In 200
the 4-day test, we found that ATV:ELQ-300 combination therapy increased the potency and 201
efficacy of treatment relative to ATV monotherapy, with ED50 values of 0.02 mg/kg and 0.01 202
mg/kg for the 1:1 and 3:1 formulations, respectively. In both ATV:ELQ-300 groups, four daily 203
doses of 1 mg/kg were fully curative. Comparison values for ATV and ELQ-300 were consistent 204
with previous reports. 205
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In the 1-day test, we also found evidence for increased activity of the combination therapy 207
regimen relative to single compound treatment. In this model, the 3:1 ratio was most effective 208
and cured mice with a single dose of 1 mg/kg, while the 1:1 combination was curative at a 209
higher, 10 mg/kg dose. Although ELQ-300 monotherapy did not prevent recrudescence in this 210
test at any dose up to its solubility limit (20 mg/kg), ATV demonstrated unanticipated single 211
dose curative activity at a dose of 10 mg/kg. More surprisingly, the 1-day ED50 and 212
nonrecrudescence values for ATV closely matched those of the 4-day treatment study, 213
suggesting that the maximum effect of this compound was obtained following a single oral dose. 214
215
In order to determine how ATV:ELQ-300 combination therapy compared to the current standard 216
of care, we next evaluated an ATV:proguanil co-formulation at the 5:2 ratio that is used in 217
Malarone (Figure 1A). In both 1-day and 4-day suppressive tests, ATV:proguanil was less 218
potent than ATV:ELQ-300 combination therapy (Table 1), although it demonstrated single dose 219
curative activity similar to that of the 3:1 ATV:ELQ-300 formulation, with a nonrecrudescence 220
dose of 1 mg/kg. Like ATV monotherapy, there was little observed benefit of multi-day dosing 221
in this model. 222
223
Acute Infection Model 224
In order to determine if ATV:ELQ-300 combination therapy was capable of clearing established 225
infections, we next tested these compounds in an acute infection model (Figures 2, 3). 226
Experimental setup was identical to suppressive tests, but treatment was delayed until initial 227
parasitemia ranged from 15-20%. Animals received either a single oral drug dose at the 228
maximum soluble concentration (20 mg/kg) or sequential 10 mg/kg doses delivered daily for 4 229
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days. Parasite clearance was monitored via daily blood smear. Although none of the tested 230
compounds were reliably curative in the single dose groups, we found that all formulations 231
containing ELQ-300 effectively prevented recrudescence in the 4-day dosing studies (Figures 232
2D, 3D, 3E). In contrast, animals treated with ATV or ATV:proguanil recrudesced and, as in the 233
suppressive tests, there was little discernable difference between single day and multi-day dosing 234
for these compounds (Figures 2A, 2C, 3C, 3F). 235
236
Resistance Propensity 237
Due to the high parasite burden at the time of drug treatment, our acute infection model also 238
served as a measure of in vivo resistance propensity. In order to determine the susceptibility of 239
recrudescent parasites to the treatment compounds, all animals were re-challenged with a 20 240
mg/kg drug dose when recrudescent parasitemia reached 15-25%. If there was any delay in 241
parasite clearance as compared to the initial drug exposure, animals were sacrificed and parasites 242
were collected for cytochrome b sequencing. We found that three ATV-treated animals harbored 243
resistant infections (Figures 2A, 2C), each with Y268C mutations corresponding to the Qo site 244
of cyt bc1 (16) (Figures 4A, 4C). Similarly, one ELQ-300 treated animal failed to respond to re-245
challenge (Figure 2B) and sequencing revealed an I22L mutation at the cyt bc1 Qi site (Figures 246
4B, 4C), which was consistent with known ELQ-300R mutations in P. falciparum (17). No 247
resistant parasites were identified in animals receiving either ATV:proguanil or ATV:ELQ-300 248
combination therapy (Figures 3A, 3F). To determine long-term potential for recrudescence, 249
animals were intermittently monitored by blood smear for at least 6 months following initial 250
infection: with the exception of the animals harboring resistant parasites (described above), the 251
re-challenge dose effectively prevented recrudescence across all treatment groups. 252
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Although ATVR and ELQ-300R strains have been well characterized in P. falciparum in vitro, we 253
next wanted to assess the degree of in vivo resistance conferred by these P. yoelii mutants. We 254
used the ELQ-300R and ATVRB strains to infect naïve mice and then performed 4-day 255
suppressive tests as described above. Although both the ATVR and ELQ-300R strains were 256
highly resistant to their respective compounds, we found that ATVR parasites remained fully 257
sensitive to ELQ-300, and vice versa (Table 2). 258
259
Discussion 260
The therapeutic combination of ATV and ELQ-300 is the first example of dual-site, antimalarial 261
inhibition of a single protein target. In P. yoelii murine models, this co-formulation was more 262
effective than ATV alone in both 1-day and 4-day suppressive tests, and the 3:1 ratio of 263
ATV:ELQ-300 was especially useful as a single-dose, blood-stage therapeutic. In comparison to 264
ATV and proguanil, which are the active components of Malarone, the ATV:ELQ-300 265
formulation demonstrated superior multi-dose activity in an acute infection model and was 266
equally effective at preventing the emergence of drug-resistant parasites. 267
268
One of our most intriguing results was that ATV monotherapy effectively cured P. yoelii 269
infected animals in our 1-day suppressive tests. Although ATV has been studied in rodents since 270
the time of its discovery in the late 1980s, the only hint of this single dose activity in the 271
literature comes from the landmark Hudson et al. paper where it was shown that 1-day and 7-day 272
ATV dosing models had similar ED50 values in P. yoelii suppressive tests (30). Our data builds 273
on that work to show that ATV is also single-dose curative in the suppressive model, and that 274
this property is retained by ATV-containing combination therapies including ATV:proguanil and 275
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ATV:ELQ-300. Although none of our tested therapies was an effective single dose cure in the 276
acute infection model, recrudescent animals re-challenged with a second 20 mg/kg dose of ELQ-277
300, ATV:proguanil, or the ATV:ELQ-300 combination therapy were permanently cleared of 278
parasites, suggesting that these formulations may be effective with a reduced or weekly dosing 279
schedule, even in acute infections. While recent clinical studies have explored the potential for 280
weekly prophylactic dosing with Malarone (31), none have yet evaluated non-daily therapy in an 281
acute setting. If effective, such regimens could dramatically reduce treatment cost and make cyt 282
bc1 inhibitors more accessible for use in endemic areas. 283
284
While the single-dose efficacy of ATV may simply be a reflection of its exceptional intrinsic 285
potency, which exceeds that of ELQ-300 by approximately 10-fold, these findings could also be 286
explained by the mechanics of the cyt bc1 complex. In yeast and bacterial model systems, 287
numerous bypass reactions partially compensate for Qi site blockade by passing electrons to 288
alternative carriers such as oxygen (32). Because similar pathways play a more minor role in Qo 289
site inhibition, it is possible that Qo site blockade more rapidly disrupts mitochondrial electron 290
transport, and hence pyrimidine biosynthesis. In contrast, the superior multi-day efficacy of 291
ELQ-300 may be attributable to the lower resistance propensity associated with this compound 292
(17). Because we only sequenced cytochrome b in highly ATV-resistant parasites, it is possible 293
that our cases of ATV recrudescence actually represented the emergence of parasites with low-294
grade ATV resistance similar to those previously reported both in vitro and in vivo (16, 33). 295
296
One lingering question is whether there is any inherent clinical risk in utilizing partner drugs 297
with the same biological target. Although this is a novel strategy for malaria, it has been used 298
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successfully in anti-cancer therapies (6). With respect to resistance, although our group has in 299
fact identified resistant P. falciparum strains in vitro with near-complete resistance to all cyt bc1 300
inhibitors (34), such mutations have never been detected in animal models or humans. These 301
resistant isolates also remain susceptible to ATV:proguanil and ELQ-300:proguanil therapy in 302
vitro, suggesting that ATV:ELQ-300:proguanil triple therapy could be a second-line strategy in 303
the unlikely event of ATV:ELQ-300 resistance. In light of the recent finding that ATVR 304
mutations prevent parasite transmission at the mosquito stage (26), we are also evaluating the 305
transmissibility of ELQ-300R parasites to determine if impaired replication within the mosquito 306
midgut is a generalizable feature of cytochrome b mutations. 307
308
Obviously, another limitation of this work is the presumption that our observed effects on P. 309
yoelii parasites may be generalized to P. falciparum and, by extension, to human disease. ELQ-310
300 has been studied in SCID mouse models, where efficacy against P. falciparum closely 311
matched that of our P. yoelii studies (18). Furthermore, the resistant mutants isolated from our P. 312
yoelii acute infection model consistently demonstrate ATVR and ELQ-300R resistance mutations 313
identified in P. falciparum, including the clinically-relevant ATVRY268C mutation noted in human 314
patients. In contrast to in vitro studies, where ELQ-300 resistance requires precise drug titration 315
(17) and ATV resistance fails to replicate the clinically-relevant mutations noted in human 316
disease (33), our model generated key resistance mutations in the span of days, suggesting that 317
our acute infection model is both a highly accurate and time-efficient tool for the evaluation of 318
antimalarial drug resistance. Ultimately, our work demonstrates that ATV:ELQ-300 is a 319
promising combination drug therapy that combines the single-dose efficacy of ATV with the low 320
resistance propensity of ELQ-300, while maintaining the remarkable potency and multi-stage 321
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activity that make cyt bc1 inhibitors an ideal choice for both prophylactic and acute antimalarial 322
therapy. 323
324
Acknowledgements 325
Competing Interests: MKR, JXK, RWW, and AN are named as co-inventors on US patent 326
2014/00458888 related to this work. Other authors have no competing interests. Data and 327
Materials Availability: ATVR and ELQ-300R P. yoelii strains will be banked with the MR4 328
malaria resource repository. 329
330
References 331
1. World Health Organization. 2015. World Malaria Report 2015. 332
2. Sibley CH. 2014. Understanding drug resistance in malaria parasites: basic science for 333
public health. Mol Biochem Parasitol 195:107-114. 334
3. Bloland P. 2001. Drug resistance in malaria. World Health Organization, Department of 335
communicable disease surveillance and response. 336
4. Winzeler E, Manary M. 2014. Drug resistance genomics of the antimalarial drug 337
artemisinin. Genome Biology 15. 338
5. Wells TN, Hooft van Huijsduijnen R, Van Voorhis WC. 2015. Malaria medicines: a 339
glass half full? Nature reviews. Drug discovery 14:424-442. 340
6. Jia J, Zhu F, Ma X, Cao Z, Li Y, Chen YZ. 2009. Mechanisms of drug combinations: 341
interaction and network perspectives. Nature reviews. Drug discovery 8:111-128. 342
7. Fischbach MA. 2011. Combination therapies for combating antimicrobial resistance. 343
Current opinion in microbiology 14:519-523. 344
on April 8, 2018 by guest
http://aac.asm.org/
Dow
nloaded from
16
8. Stickles AM, Ting LM, Morrisey JM, Li Y, Mather MW, Meermeier E, Pershing 345
AM, Forquer IP, Miley GP, Pou S, Winter RW, Hinrichs DJ, Kelly JX, Kim K, 346
Vaidya AB, Riscoe MK, Nilsen A. 2015. Inhibition of cytochrome bc1 as a strategy for 347
single-dose, multi-stage antimalarial therapy. The American journal of tropical medicine 348
and hygiene 92:1195-1201. 349
9. Srivastava IK, Rottenberg H, Vaidya AB. 1997. Atovaquone, a broad spectrum 350
antiparasitic drug, collapses mitochondrial membrane potential in a malarial parasite. 351
Journal of Biological Chemistry 272:3961-3966. 352
10. Nilsen A, Miley GP, Forquer IP, Mather MW, Katneni K, Li Y, Pou S, Pershing 353
AM, Stickles AM, Ryan E, Kelly JX, Doggett JS, White KL, Hinrichs DJ, Winter 354
RW, Charman SA, Zakharov LN, Bathurst I, Burrows JN, Vaidya AB, Riscoe MK. 355
2014. Discovery, synthesis, and optimization of antimalarial 4(1H)-Quinolone-3-356
Diarylethers. Journal of medicinal chemistry 57:3818-3834. 357
11. Painter HJ, Morrisey JM, Mather MW, Vaidya AB. 2007. Specific role of 358
mitochondrial electron transport in blood-stage Plasmodium falciparum. Nature 446:88-359
91. 360
12. Vaidya AB, Mather MW. 2009. Mitochondrial evolution and functions in malaria 361
parasites. Annual review of microbiology 63:249-267. 362
13. Looareesuwam S, Chulay JD, Canfield CJ, Hutchinson DBA. 1999. Malarone 363
(atovaquone and proguanil hydrochloride): A review of its clinical development for 364
treatment of malaria. The American journal of tropical medicine and hygiene 60:533-365
541. 366
on April 8, 2018 by guest
http://aac.asm.org/
Dow
nloaded from
17
14. Birth D, Kao WC, Hunte C. 2014. Structural analysis of atovaquone-inhibited 367
cytochrome bc1 complex reveals the molecular basis of antimalarial drug action. Nature 368
communications 5:4029. 369
15. Fisher N, Abd Majid R, Antoine T, Al-Helal M, Warman AJ, Johnson DJ, 370
Lawrenson AS, Ranson H, O'Neill PM, Ward SA, Biagini GA. 2012. Cytochrome b 371
mutation Y268S conferring atovaquone resistance phenotype in malaria parasite results in 372
reduced parasite bc1 catalytic turnover and protein expression. The Journal of biological 373
chemistry 287:9731-9741. 374
16. Vaidya AB, Mather MW. 2000. Atovaquone resistance in malaria parasites. Drug 375
resistance updates : reviews and commentaries in antimicrobial and anticancer 376
chemotherapy 3:283-287. 377
17. Stickles AM, de Almeida MJ, Morrisey JM, Sheridan KA, Forquer IP, Nilsen A, 378
Winter RW, Burrows JN, Fidock DA, Vaidya AB, Riscoe MK. 2015. Subtle changes 379
in endochin-like quinolone structure alter the site of inhibition within the cytochrome bc1 380
complex of Plasmodium falciparum. Antimicrobial agents and chemotherapy 59:1977-381
1982. 382
18. Nilsen A, LaCrue AN, White KL, Forquer IP, Cross RM, Marfurt J, Mather MW, 383
Delves MJ, Shackleford DM, Saenz FE, Morrisey JM, Steuten J, Mutka T, Li Y, 384
Wirjanata G, Ryan E, Duffy S, Kelly JX, Sebayang BF, Zeeman AM, Noviyanti R, 385
Sinden RE, Kocken CH, Price RN, Avery VM, Angulo-Barturen I, Jimenez-Diaz 386
MB, Ferrer S, Herreros E, Sanz LM, Gamo FJ, Bathurst I, Burrows JN, Siegl P, 387
Guy RK, Winter RW, Vaidya AB, Charman SA, Kyle DE, Manetsch R, Riscoe MK. 388
on April 8, 2018 by guest
http://aac.asm.org/
Dow
nloaded from
18
2013. Quinolone-3-diarylethers: a new class of antimalarial drug. Science translational 389
medicine 5:177ra137. 390
19. Edstein MD, Kotecka BM, Anderson KL, Pombo DJ, Kyle DE, Rieckmann KH, 391
Good MF. 2005. Lengthy antimalarial activity of atovaquone in human plasma following 392
atovaquone-proguanil administration. Antimicrobial agents and chemotherapy 49:4421-393
4422. 394
20. Pimentel S, Nogueira F, Benchimol C, Quinhentos V, Bom J, Varandas L, do 395
Rosario V, Bernardino L. 2006. Detection of atovaquone-proguanil resistance 396
conferring mutations in Plasmodium falciparum cytochrome b gene in Luanda, Angola. 397
Malaria journal 5:30. 398
21. Plucinski MM, Huber CS, Akinyi S, Dalton W, Eschete M, Grady K, Silva-Flannery 399
L, Mathison BA, Udhayakumar V, Arguin PM, Barnwell JW. 2014. Novel mutation 400
in cytochrome b of Plasmodium falciparum in one of two atovaquone-proguanil 401
treatment failures in travelers returning from same site in Nigeria. Open forum infectious 402
diseases 1:ofu059. 403
22. Jones K, Ward SA. 2002. Biguanide-atovaquone synergy against Plasmodium 404
falciparum in vitro. Antimicrobial agents and chemotherapy 46:2700-2703. 405
23. Srivastava IK, Vaidya AB. 1999. A mechanism for the synergistic antimalarial action of 406
atovaquone and proguanil. Antimicrobial agents and chemotherapy 43:1334-1339. 407
24. Vaidya A. 2012. Naphthoquinones: atovaquone and other antimalarials targeting 408
mitochondrial functions. Milestones in drug therapy. Treatment and prevention of 409
malaria: Antimalarial drug chemistry, action and use:127-139. 410
on April 8, 2018 by guest
http://aac.asm.org/
Dow
nloaded from
19
25. Maude RJ, Nguon C, Dondorp AM, White LJ, White NJ. 2014. The diminishing 411
returns of atovaquone-proguanil for elimination of Plasmodium falciparum malaria: 412
modelling mass drug administration and treatment. Malaria 13. 413
26. Goodman CD, Siregar JE, Mollard V, Vega-Rodriguez J, Syafruddin D, Matsuoka 414
H, Matsuzaki M, Toyama T, Sturm A, Cozijnsen A, Jacobs-Lorena M, Kita K, 415
Marzuki S, McFadden GI. 2016. Parasites resistant to the antimalarial atovaquone fail 416
to transmit by mosquitoes. Science 352:349-353. 417
27. Fivelman QL, Adagu IS, Warhurst DC. 2004. Modified fixed-ratio isobologram 418
method for studying in vitro interactions between atovaquone and proguanil or 419
dihydroartemisinin against drug-resistant strains of Plasmodium falciparum. 420
Antimicrobial agents and chemotherapy 48:4097-4102. 421
28. Smilkstein M, Sriwilaijaroen N, Kelly JX, Wilairat P, Riscoe MK. 2004. Simple and 422
inexpensive fluorescence-based technique for high-throughput antimalarial drug 423
screening. Antimicrob Agents Chemotherapy 48:1803-1806. 424
29. Malleret B, Claser C, Ong AS, Suwanarusk R, Sriprawat K, Howland SW, Russell 425
B, Nosten F, Renia L. 2011. A rapid and robust tri-color flow cytometry assay for 426
monitoring malaria parasite development. Scientific reports 1:118. 427
30. Hudson AT, Dickins M, Ginger CD, Gutteridge WE, Holdich T, Hutchinson DBA, 428
Pudney M, Randall AW, Latter VS. 1991. 566C80: A potent broad spectrum anti-429
infective agent with activity against malaria and opportunistic infections in AIDS 430
patients. Drugs Under Experimental and Clinical Research 17:427-435. 431
31. Deye GA, Miller RS, Miller L, Salas CJ, Tosh D, Macareo L, Smith BL, Fracisco S, 432
Clemens EG, Murphy J, Sousa JC, Dumler JS, Magill AJ. 2012. Prolonged protection 433
on April 8, 2018 by guest
http://aac.asm.org/
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nloaded from
20
provided by a single dose of atovaquone-proguanil for the chemoprophylaxis of 434
Plasmodium falciparum malaria in a human challenge model. Clinical infectious diseases 435
: an official publication of the Infectious Diseases Society of America 54:232-239. 436
32. Muller F, Crofts AR, Kramer DM. 2002. Multiple Q-cycle bypass reactions at the Qo 437
site of the cytochrome bc1 complex. Biochemistry 41:7866-7874. 438
33. Schwobel B, Alifrangis M, Salanti A, Jelinek T. 2003. Different mutation patterns of 439
atovaquone resistance to Plasmodium falciparum in vitro and in vivo: Rapid detection of 440
codon 268 polymorphisms in the cytochrome b as potential in vivo resistance marker. 441
Malaria journal 2. 442
34. Smilkstein MJ, Forquer I, Kanazawa A, Kelly JX, Winter RW, Hinrichs DJ, 443
Kramer DM, Riscoe MK. 2008. A drug-selected Plasmodium falciparum lacking the 444
need for conventional electron transport. Mol Biochem Parasitol 159:64-68. 445
446
Figure Legends 447
Figure 1: Structures of test compounds (A) and Isobolar analysis of ATV and ELQ-300 in 448
vitro (B). ATV and ELQ-300 are moderately synergistic in vitro with a mean FIC index of 0.65. 449
Data represent mean FICs of ATV and ELQ-300 as assessed in D6 P. falciparum parasites. Data 450
derived from two independent experiments, error bars = S.E.M. Dotted line represents a 451
comparative FIC index of 1. 452
453
Figure 2: Efficacy of ATV and ELQ-300 in murine acute infection models. In both 1-day (A) 454
and 4-day (C) treatment models, ATV therapy was associated with rapid parasite clearance, but 455
high resistance propensity. In contrast, while ELQ-300 treated animals recrudesced in 1-day 456
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models (B), 4-day treatment (D) universally prevented recrudescence. Grey bars/circles indicate 457
drug administration. Arrows indicate animals sacrificed for blood collection and parasite gene 458
sequencing. Each trace represents a single animal, N=4 animals/group 459
460
Figure 3: Comparison of ATV:ELQ-300 and ATV:proguanil combination therapies in 461
murine acute infection models. In 1-day studies, ATV:ELQ-300 combination therapy (A, B) 462
delayed recrudescence as compared to ATV or ELQ-300 comparison groups, and one animal in 463
the 3:1 ATV:ELQ-300 group (B) was fully cured with a single 20 mg/kg oral dose. In 4-day 464
tests, ATV:ELQ-300 combination therapy effectively prevented recrudescence when delivered as 465
either 1:1 (D) or 3:1 (E) co-formulations. No resistant parasites were detected in any ATV:ELQ-466
300 treatment group. In contrast, while ATV:proguanil effectively prevented resistance, it did 467
not delay the onset of recrudescence in either 1-day (C) or 4-day (F) treatment groups as 468
compared to ATV monotherapy. Grey bars/circles represent drug administration. Arrows 469
indicate animals sacrificed for blood collection and parasite gene sequencing. Each trace 470
represents a single animal, N=4 animals/group. 471
472
Figure 4: Sequence alignments of cytochrome b in ELQ-300R and ATVR P. yoelii parasites. 473
Three strains collected from ATV-treated animals in the 1-day acute treatment model (ATVR A, 474
ATVR B, and ATVR C) contained Y268C mutations at the cyt bc1 QO site (A), while parasites 475
isolated following ELQ-300 exposure contained an I22L mutation at the cyt bc1 Qi site (B). 476
Parasites sequenced from ATV:ELQ-300 and ATV:proguanil treated animals matched wild-type. 477
Schematic of relevant Qo and Qi site residues as well as the Y268S and I22L mutations 478
characteristic of ATV and ELQ-300 resistance shown in figure (C). 479
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Tables 480
Treatment
4-Day Dosing (mg/kg)
1-Day Dosing (mg/kg)
ED50
NRD ED50 NRD
ELQ-300 0.02 0.3 0.03 >20 ATV 0.1 10 0.08 10
(1:1) ATV:300 0.02 1 0.1 10 (3:1) ATV:300 0.01 1 0.14 1 (5:2) ATV:PG 0.14 1 0.5 1*
481
Table 1: Comparative in vivo activities of ELQ-300, ATV, and combination therapies in the 482
murine Peters suppressive test. Co-formulation of ATV with either ELQ-300 or proguanil 483
increased in vivo efficacy in both 4-day and 1-day studies. ATV, ATV:ELQ-300, and 484
ATV:proguanil were all capable of clearing parasites with a single oral dose. ED50 = 50% 485
effective dose, the dose required to suppress day 5 parasitemias by 50% relative to untreated 486
controls. NRD = non-recrudescence or curative dose, dose required to clear parasites from 487
bloodstream for 30 days following infection. ATV = atovaquone, PG = proguanil. 300 = ELQ-488
300. * = ¾ animals cured at 1 mg/kg ATV:PG. N=4 animals/group 489
490
491
492
493
494
495
496
497
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Strain Atovaquone (mg/kg/day) ELQ-300 (mg/kg/day) ED50 NRD ED50 NRD P. yoelii wild type 0.03 1.0 0.02 1.0
P. yoelii (ATVRY268C) >10 >10 0.02 1.0
P. yoelii (ELQ-300RI22L) 0.03 1.0 1.0 >10
498
Table 2: Susceptibility of ATVR and ELQ-300R mutants isolated from the acute treatment 499
model to ELQ-300 and ATV in 4-day suppressive tests. In both cases, mutations conferred 500
high-grade resistance to the parent compound, but parasites remained fully sensitive to inhibition 501
at the alternate site of cyt bc1. 10 mg/kg represents the solubility limit of ELQ-300 in 100 μL of 502
the PEG-400 vehicle. N=4 animals/group. 503
504
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0.0 0.5 1.00.0
0.5
1.0
FIC ELQ-300
FIC
AT
V
ATV 300 Isobol.pzf:Layout 1 - Wed Dec 31 16:55:48 1969
A B
A B
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ATV Single Dose(20 mg/kg)
0 5 10 150
50
100
150
60
Post-Treatment Day
Pe
rce
nt
of
Pre
-Tre
atm
en
t
Para
sit
em
ia
ELQ-300 Single Dose(20 mg/kg)
0 5 10 150
50
100
60
Post-Treatment Day
Pe
rce
nt
of
Pre
-Tre
atm
en
t
Para
sit
em
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ATV Multi Dose(10 mg/kg x 4)
0 5 10 150
50
100
150
60
Post-Treatment Day
Perc
en
t o
f P
re-T
rea
tmen
t
Para
sit
em
ia
ELQ-300 Multi Dose(10 mg/kg x 4)
0 5 10 150
50
100
60
Post-Treatment Day
Pe
rce
nt
of
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-Tre
atm
en
t
Para
sit
em
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ATV 300 Combo.pzf:Layout 1 - Sun Sep 27 16:15:34 2015
A
C
B
D
ATVR A
ELQ-300R
ATVR B
ATVR C
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ATV:ELQ-300 (1:1)Single Dose (20 mg/kg)
0 5 10 150
50
100
60
Post-Treatment Day
Pe
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atm
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ATV:ELQ-300 (3:1)Single Dose (20 mg/kg)
0 5 10 150
50
100
150
60
Post-Treatment Day
Pe
rcen
t o
f P
re-T
rea
tme
nt
Para
sit
em
ia
ATV:Proguanil (5:2)Single Dose (20 mg/kg)
0 5 10 150
50
100
150
60
Post-Treatment Day
Pe
rce
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Pre
-Tre
atm
en
t
Para
sit
em
iaATV:ELQ-300 (1:1)
Multi-Dose (10 mg/kg x 4)
0 5 10 150
50
100
60
Post-Treatment Day
Perc
en
t o
f P
re-T
reatm
en
t
Para
sit
em
ia
ATV:ELQ-300 (3:1)Multi Dose (10 mg/kg x 4)
0 5 10 150
50
100
60
Post-Treatment Day
Pe
rce
nt
of
Pre
-Tre
atm
en
t
Para
sit
em
ia
ATV:Proguanil (5:2)Multi Dose (10 mg/kg x 4)
0 5 10 150
50
100
150
60
Post-Treatment Day
Pe
rce
nt
of
Pre
-Tre
atm
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t
Para
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ATV 300 Combo.pzf:Layout 2 - Sun Sep 27 16:15:48 2015
A B C
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4
A
Query 241 SHPDNAIIVNTYVTPLQIVPEWYFLPFYAMLKTIPSKNAGLVIVVASLQLLFLLAEQRNL 300
SHPDNAIIVNTYVTPLQIVPEWYFLPF+AMLKTIPSKNAGLVIVVASLQLLFLLAEQRNL
Subject 241 SHPDNAIIVNTYVTPLQIVPEWYFLPFCAMLKTIPSKNAGLVIVVASLQLLFLLAEQRNL 300
B
Query 1 MNYNSINLVKTHLINYPCPLNINFLWNYGFLLGIIFFIQILTGVFLASRYSPEISYAYYS 60
MNYNSINLVKTHLINYPCPLN+NFLWNYGFLLGIIFFIQILTGVFLASRYSPEISYAYYS
Subject 1 MNYNSINLVKTHLINYPCPLNLNFLWNYGFLLGIIFFIQILTGVFLASRYSPEISYAYYS 60
C
I22L
Matrix
Qi site
Y268C
Intermembrane space
QO site
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