Nilsen 1..13

Quinolone-3-Diarylethers: A New Class of Antimalarial Drug Aaron Nilsen et al. Sci Transl Med 5, 177ra37 (2013); DOI: 10.1126/scitranslmed.3005029

Editor's Summary

Taking the Bite Out of Malaria

Like quinine, most antimalarial drugs in use today target only the symptomatic blood stage. The efficacy of these drugs has been compromised by resistance, and so there is a pressing need for new drugs that target multiple stages of the parasite life cycle for use in malaria treatment and prevention. Clearly, it is advantageous to strike at the liver stage where parasite numbers are low, to diminish the likelihood of selecting for a resistant mutant and before the infection has a chance to weaken the defenses of the human host. In a new study, Nilsen and colleagues describe ELQ-300, a 4(1H)-quinolone-3-diarylether, which targets the liver and blood stages, including the forms that are crucial to disease transmission (gametocytes, zygotes, and ookinetes). In mouse models of malaria, a single oral dose of 0.03 mg/kg prevented sporozoite-induced infections, whereas four daily doses of 1 mg/kg achieved complete cures of patent infections. ELQ-300 is a preclinical candidate that may be coformulated with other antimalarials to prevent and treat malaria, with the potential to aid in eradication of the disease.

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Malaria is spread from person to person by mosquitoes that inject 8 to 10 sporozoite forms of the parasite in a single bite. The sporozoites reproduce in the liver to produce 10,000 to 30,000 merozoites before the liver schizont ruptures and parasites flood into the bloodstream where the absolute parasite burden may increase to a thousand billion (10 12) circulating parasites. Some of these parasites develop into gametocytes that may be ingested by another mosquito where they progress through ookinete, oocyst, and sporozoite stages to complete the cycle.

RESEARCH ARTICLE MALARIA

Quinolone-3-Diarylethers: A New Class of Antimalarial Drug

The goal for developing new antimalarial drugs is to find a molecule that can target multiple stages of the parasite’s life cycle, thus impacting prevention, treatment, and transmission of the disease. The 4(1H)-quinolone-3diarylethers are selective potent inhibitors of the parasite’s mitochondrial cytochrome bc1 complex. These compounds are highly active against the human malaria parasites Plasmodium falciparum and Plasmodium vivax. They target both the liver and blood stages of the parasite as well as the forms that are crucial for disease transmission, that is, the gametocytes, the zygote, the ookinete, and the oocyst. Selected as a preclinical candidate, ELQ-300 has good oral bioavailability at efficacious doses in mice, is metabolically stable, and is highly active in blocking transmission in rodent models of malaria. Given its predicted low dose in patients and its predicted long half-life, ELQ-300 has potential as a new drug for the treatment, prevention, and, ultimately, eradication of human malaria.

INTRODUCTION Malaria remains the most significant parasitic disease in the tropics where it causes ~200 million clinical cases and is reported to claim up to 1.2 million lives each year (1). Most of the drugs used to treat the disease target only the blood stages of infection, and the efficacy of many of the traditional antimalarials, such as chloroquine, has been declining because of the emergence of multidrug-resistant strains of Plasmodium parasites. The widespread nature of multidrug resistance has shifted the emphasis to artemisinin combination therapies (ACTs) 1 Veterans Affairs Medical Center, 3710 Southwest U.S. Veterans Hospital Road, Portland, OR 97239, USA. 2Department of Global Health, College of Public Health, 3720 Spectrum Boulevard (Suite 304), Tampa, FL 33612, USA. 3Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia. 4Department of Chemistry, University of South Florida, 4202 East Fowler Avenue, Tampa, FL 33620–5250, USA. 5Global and Tropical Health Division, Menzies School of Health Research and Charles Darwin University, PO Box 41096, Casuarina, NT 0811, Australia. 6 Department of Microbiology and Immunology, Drexel University College of Medicine, 2900 Queen Lane, Philadelphia, PA 19129, USA. 7Department of Life Sciences, Imperial College London, London SW7 2AZ, UK. 8Eskitis Institute for Cell and Molecular Therapies, Brisbane Innovation Park, Nathan Campus, Griffith University, Brisbane, Queensland 4111, Australia. 9Eijkman Institute for Molecular Biology, Jl. Diponegoro 69, Jakarta 10430, Indonesia. 10Department of Parasitology, Biomedical Primate Research Centre, P. O. Box 3306, 2280 GH Rijswijk, the Netherlands. 11Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 7LJ, UK. 12GlaxoSmithKline, Medicines Development Campus, Diseases of the Developing World, Severo Ochoa 2, Tres Cantos 28760, Madrid, Spain. 13Medicines for Malaria Venture, 20, route de Pré-Bois, P. O. Box 1826, 1215 Geneva 15, Switzerland. 14Siegl Pharma Consulting LLC, Blue Bell, PA 19422, USA. 15 Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105–3678, USA. 16Department of Molecular Microbiology and Immunology, 3181 Sam Jackson Boulevard, Portland, OR 97239, USA. *These authors contributed equally to this work. †Present address: Centro de Investigación en Enfermedades Infecciosas, Escuela de Ciencias Biológicas, Pontificia Universidad Católica del Ecuador, 12 de Octubre 1076 y Roca, Quito, Ecuador. ‡Corresponding author. E-mail: [email protected] (M.K.R.); [email protected] (R.M.)

as first-line treatments in malaria-endemic countries. Whereas ACTs have succeeded in reducing mortality, and mosquito control programs have helped to reduce the worldwide burden of disease, there are warning signs of emerging artemisinin resistance in Southeast Asia (2, 3). As a result, the development of new drugs for the treatment and prevention of malaria is urgently needed to circumvent resistance and to meet the challenge of malaria eradication. To support the global malaria eradication effort, there is a need for new medicines that can be given by mouth as a single dose to allow direct monitoring of administration and to ensure compliance (4, 5). Ideally, such drugs would be efficacious against liver- and blood-stage infections and active against resistant strains. There is also a need for next-generation drugs that kill gametocytes as well as the vector stages (that is, ookinetes and oocysts) and thus can be used to prevent disease transmission. These desirable features would need to be incorporated into new molecules with longer half-lives for chemoprophylaxis and to provide long-term protection against reinfection. Several years ago, the Medicines for Malaria Venture formed a team of investigators to evaluate the potential for development of an endochin derivative for clinical use (Fig. 1). The team used a test cascade to optimize endochin, which included the design and chemical synthesis of structural analogs, in vitro tests to compare antiplasmodial activity and mammalian cytotoxicity with profiling of the life cycle fingerprint across all parasite stages, assessment of ex vivo efficacy against Plasmodium falciparum and Plasmodium vivax clinical field isolates, the determination of metabolic stability and enzyme inhibition, assessment of in vivo efficacy against blood and liver stages of infection in murine models of malaria, and pharmacokinetic and safety profiling. Here, we report on two 4(1H)-quinolone derivatives, ELQ-300 and P4Q-391, that were identified as late lead candidates.

www.ScienceTranslationalMedicine.org

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Vol 5 Issue 177 177ra37

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Aaron Nilsen,1* Alexis N. LaCrue,2* Karen L. White,3 Isaac P. Forquer,1 R. Matthew Cross,4 Jutta Marfurt,5 Michael W. Mather,6 Michael J. Delves,7 David M. Shackleford,3 Fabian E. Saenz,2† Joanne M. Morrisey,6 Jessica Steuten,3 Tina Mutka,2 Yuexin Li,1 Grennady Wirjanata,5 Eileen Ryan,3 Sandra Duffy,8 Jane Xu Kelly,1 Boni F. Sebayang,9 Anne-Marie Zeeman,10 Rintis Noviyanti,9 Robert E. Sinden,7 Clemens H. M. Kocken,10 Ric N. Price,5,11 Vicky M. Avery,8 Iñigo Angulo-Barturen,12 María Belén Jiménez-Díaz,12 Santiago Ferrer,12 Esperanza Herreros,12 Laura M. Sanz,12 Francisco-Javier Gamo,12 Ian Bathurst,13 Jeremy N. Burrows,13 Peter Siegl,14 R. Kiplin Guy,15 Rolf W. Winter,1 Akhil B. Vaidya,6 Susan A. Charman,3 Dennis E. Kyle,2 Roman Manetsch,4‡ Michael K. Riscoe1,16‡

RESEARCH ARTICLE

Fig. 1. Chemical structures of the compounds used or referred to in this study. Atovaquone and GW844520 are antimalarial drugs that are known to target the Plasmodium cytochrome bc1 complex. Discovery of endochin as an antimalarial dates back to the 1940s when it was shown to be effective against several avian plasmodial species but lacked activity against mammalian species. A new structural class of antimalarial agents is represented by ELQ-271, ELQ-300, and P4Q-391, 4(1H)-quinolones bearing a diarylether side chain at the 3 position of the quinolone core. ELQ-300 and P4Q-391 were developed further and tested in a number of rodent models of malaria.

Salzer and co-workers first described endochin more than 70 years ago (6). The drug attracted interest because of its ability to prevent and treat sporozoite-induced malaria in an avian model of the disease (Plasmodium cathemerium in canaries), but it failed to work in humans (7). More recent studies have confirmed that endochin exhibits potent antiplasmodial activity. However, it is metabolically unstable in the presence of liver microsomes from all tested mammalian species, including human, and is converted to poorly active metabolites (8, 9). As a consequence, we set out to design endochin derivatives with enhanced metabolic stability and potent activity against drug-resistant malaria parasites. Endochin appears to target parasite mitochondrial processes as it is known to block respiration in Plasmodium-infected red blood cells (9). The parasite mitochondrion has been validated as a target for malaria therapy by the clinical success of the drug atovaquone (Fig. 1), which selectively inhibits the parasite cytochrome bc1 complex (10). Because atovaquone monotherapy leads to the rapid selection of drugresistant mutants, it is co-administered with proguanil in a synergistic combination (Malarone). Although Malarone is commonly prescribed for malaria prophylaxis in travel medicine, it is not widely used in malaria control programs because of the high cost of production. As a result, there is increasing interest in the development of less expensive alternatives to atovaquone that target parasite respiration, are active against all known strains, and are not prone to rapid resistance selection. Yeates and co-workers recently described a new series of diphenylethersubstituted pyridones that, like atovaquone, are believed to target the Qo site of the Plasmodium cytochrome bc1 complex of the mitochondrial electron transport chain (11). A lead compound from this series, GW844520 (Fig. 1), was more potent than chloroquine for treating mice infected with the murine malaria parasite Plasmodium yoelii. A structurally related pyridone (GSK932121) was moved into clinical trials in healthy volunteers (phase 1), and low doses were delivered

RESULTS Antiplasmodial activities of ELQ-300 and P4Q-391 Antiplasmodial activities were determined by two different assays: the fluorescence SYBR Green method (15) and the [3H]hypoxanthine incorporation assay (16). ELQ-300 and P4Q-391 displayed potent activity with low nanomolar EC50 (median effective concentration) values at 72 hours against chloroquine-sensitive (D6 and 3D7) and multidrugresistant P. falciparum strains (Dd2, W2, and recent isolates from Southeast Asia) including the atovaquone-resistant clinical isolate TM90-C2B (Tables 1 and 2). Atovaquone resistance in strain TM90-C2B is linked to a mutation in the parasite’s cytochrome b gene (the mutation alters the wild type from Tyr268 to Ser268) in the ubiquinol-binding region also known as the Qo site. EC50 values for ELQ-300 against the reference strains and recent isolates range from 1.3 to 13.6 nM, whereas the range of EC50 values for P4Q-391 is slightly higher (EC50 range, 3.18 to 32.3 nM). Cross-resistance with atovaquone in the TM90-C2B strain was reduced for both ELQ-300 (1.3-fold) and P4Q-391 (3.2-fold) compared to the diarylether substituted pyridone GSK932121A (16.7-fold). We also included the transgenic P. falciparum clone D10yDHOD and A6 clone (17) to demonstrate that the quinolone-3-diarylethers target the parasite respiratory chain. D10yDHOD parasites express the Saccharomyces cerevisiae dihydroorotate dehydrogenase, which is a cytosolic enzyme that does not require ubiquinone as an electron acceptor (18, 19). This genetic manipulation uncouples pyrimidine biosynthesis from electron transport and endows these parasites with protection against electron transport inhibitors (19). As shown in Tables 1 and 2, D10yDHOD transfectants are insensitive to ELQ-300 and P4Q-391, results that are consistent with the respiratory chain as their primary site of action. We investigated the ex vivo susceptibility profile of both compounds against clinical isolates of P. falciparum and P. vivax collected from patients attending malaria clinics in southern Papua, Indonesia, a region where multidrug-resistant strains of both P. falciparum and P. vivax are endemic. Drug susceptibility was measured using a modified microtest developed by the World Health Organization (WHO) and described previously (20, 21). ELQ-300 performed well against both drug-resistant P. falciparum and P. vivax field isolates with median EC50 values of 14.9 and 17.9 nM, respectively (Fig. 2, A and B). There was no statistical difference in ex vivo susceptibility to ELQ-300 between the two Plasmodium species. Median EC50 values of 30.6 nM


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with success. However, the molecule was recently withdrawn from human trials because of safety concerns observed with a prodrug of GSK932121 in preclinical studies (12). The toxicity observed in rats was attributed to the inhibition of mammalian mitochondrial cytochrome bc1 complex by the parent drug GSK932121. Earlier work by our respective research teams focused on endochin derivatives with varying substituents in the benzenoid ring of the quinolone core (8, 9, 13). Endochin analogs displaying simple alkyl or aryl groups at the 3 position were made to increase our understanding of the antimalarial structure-activity profile (8, 9, 13). A decision was made to follow the lead from the GlaxoSmithKline (GSK) pyridone project by placing a diphenylether side chain at the critical 3 position (14). ELQ300 and P4Q-391 contain this diphenylether side chain at the 3 position. The current study examines the antiplasmodial activities and toxicity of ELQ-300 and P4Q-391 in several rodent models of malaria.

Table 1. Antiplasmodial IC50 values (nM) (SYBR Green method) for standard isolates and clones of P. falciparum. IC50 values were determined by the 72-hour SYBR Green fluorescence–based method described previously by Smilkstein et al. (15). Drug

D6

W2

Dd2

TM90-C2B

D10yDHOD

ELQ-300

2.2

1.8

2.5

1.7

>2500

P4Q-391

7.7

7.6

7.7

20.4

>2500

Atovaquone

0.1

0.3

0.1

>2500

>2500

Chloroquine

7.8

126

153

92.7

82

Table 2. Antiplasmodial IC50 values (nM) ([3H]hypoxanthine incorporation method) for isolates and clones of P. falciparum. IC50 values were determined by monitoring for the incorporation of [3H]hypoxanthine into parasite nucleic acids as described previously (16). The PL and ARC series are isolates from Cambodia obtained in 2008. The Dd2 End isolates were selected for resistance to endochin in vitro (Vaidya, unpublished observations). Pf strain

ATQ ELQ-300 P4Q-391 GSK932121A Chloroquine

W2

0.3

2.27

TM90-C2B

>170

2.84

PL08-009

0.17

PL08-025

2.18

1.32 13.6

3.18 32.3

2.57 15.5

63 59.9 262

0.17 >170

3D7 luc

0.36

4.79

12

6.45

21.9

D6

0.31

3.06

11.27

3.66

22.7

Dd2 EndR1

5.13

3.24

5.02

8.89

63.1

Dd2 EndR2

2.1

1.36

7.13

5.95

69.1

Dd2 EndR3

8.26

TM91-C235

0.56

5.99 >210 4.34

>200

2.37

215

A6

>210

4.44

3.39 56.8

ARC08-0063

D10 attb yDHOD >170

1.52

7.45 23.5

>235

38.3 21.5

11.3

14.8

106

>200

>235

21.4

12

6.25

123

(P. falciparum) and 54.5 nM (P. vivax) for P4Q-391 were somewhat higher than those for ELQ-300, and there was a statistically significant (P = 0.001) difference in ex vivo susceptibility to P4Q-391 between the two Plasmodium species. Together, our data show that ELQ-300 potently inhibits erythrocytic stages of the two most significant human malaria species, P. falciparum and P. vivax, and that it exhibits greater intrinsic antiplasmodial activity in vitro in comparison to P4Q-391. Cytotoxicity and selective inhibition of parasite cytochrome bc1 by ELQ-300 and P4Q-391 The two 4(1H)-quinolones were tested for cytotoxicity against J774 mouse macrophages, low-passage (3 to 10) human foreskin fibroblasts, and murine bone marrow–derived hematopoietic progenitor cells including CFU-GM (granulocyte-macrophage), BFU-E (erythroid), and CFU-GEMM (pluripotent) stem cells. The 50% inhibitory concentrations for ELQ-300 and P4Q-391 in each of these systems were >10 mM (table S1). Considering that the antiplasmodial EC50 values for these drugs are in the low nanomolar range, the comparative cytotoxicity

data provide an in vitro selectivity index of 400 to greater than 1000-fold. Drug safety is especially important in malaria because pregnant women and young children are the most vulnerable to the lethal consequences of infection and the most likely to require treatment. For this reason, it was important to establish that 4(1H)-quinolones could be modified to avoid inhibition of human cytochrome bc1. A clear structure-activity profile emerged for the 4(1H)-quinolone series and their inhibitory activity toward human cytochrome bc1 (derived from HEK cells). The data presented in Table 3 show a correlation between inhibition and the nature of the substitution pattern on the benzenoid ring of the quinolone core. ELQ-271 (Fig. 1), with an unsubstituted benzenoid ring, inhibits the human enzyme [median inhibitory concentration (IC50) = 1.99 mM], whereas the placement of a chlorine atom and/or a methoxy group in this ring hinders interaction with human cytochrome bc1, because IC50 values for ELQ-300 and P4Q-391 are greater than 10 mM (table S2). Previously, these same substitutions on the benzenoid ring were shown to enhance antimalarial potency of endochin derivatives in vitro (8, 9). We also conducted adenosine triphosphate (ATP) depletion assays using two different mammalian cell lines to validate the degree of selectivity that was observed in the enzyme inhibitor assays. As shown in Fig. 3, ELQ-271 caused a concentration-dependent decline in ATP levels (indicating that it was inhibiting host electron transport processes) with EC50 values in the range of 7.4 to 15.4 mM, whereas ELQ-300 did not have a measurable adverse effect on ATP levels in either cell line at concentrations of 10 mM and higher. The ability of ELQ-300 and P4Q-391 to inhibit cytochrome bc1 activity from P. falciparum mitochondria was assessed. As shown in Table 3, both compounds are potent inhibitors of the P. falciparum cytochrome bc1, with IC50 values of 0.58 and 1.06 nM, respectively (Fig. 4). Mather et al. have previously reported an IC50 value of 2.0 nM for atovaquone against P. falciparum cytochrome bc1 (22). Together, our data show that ELQ-300 and P4Q-391 are highly selective inhibitors of plasmodial cytochrome bc1 complexes with selectivity indices that are ≥10,000. This high level of selectivity suggests a low potential for side effects in humans because of inhibition of the host enzyme. Speed of action of ELQ-300, propensity to induce resistance, and synergism with proguanil Speed of action is an important determinant of antimalarial efficacy and a crucial parameter guiding clinical utility. Antimalarial endoperoxides like artesunate are rapid-acting drugs that typically clear parasitemia in the first 48 hours of treatment. In contrast, atovaquone displays an initial lag phase of cytostatic action over the first 48 hours, after which the drug exerts cidal activity (23). We used an in vitro assay to measure the effect of ELQ-300 on parasite viability over time by exposing P. falciparum to the drug for up to 120 hours (23). Our results show that ELQ-300 is a slow-acting drug with a delayed parasite reduction ratio (PRR) similar to atovaquone. The time required to kill >99.9% of parasites treated with 10× EC50 concentration of ELQ-300 is estimated to be ≈100 hours (fig. S1). Drug resistance is inevitable for antimalarial agents, and consequently, it is prudent to evaluate new candidates to ensure a low propensity for resistance development. Experiments were performed with 108 Dd2 parasites exposed to 10× EC50 of either atovaquone (10 nM) or ELQ-300 (150 nM) (table S3). Cultures were continued under drug


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RESEARCH ARTICLE

Although our studies showed that the propensity for inducing parasite resistance to both 4(1H)-quinolones was quite low relative to atovaquone, we were interested in evaluating the potential for synergism with proguanil, the clinical partner of atovaquone in Malarone. For in vitro isobolar testing of ELQ-300 and proguanil, we used fixed combination ratios of drug mixtures (24). As shown in fig. S2, the antiplasmodial effect was greater at every dose combination than expected for a simple additive response. On the basis of isobolar testing against four different P. falciparum strains (D6, Dd2, TM90C2B, and V1/S), we concluded that a strong pharmacological synergism exists between ELQ-300 and proguanil. In vivo efficacy against blood and liver stages of the parasite life cycle Efficacy against erythrocytic stages was determined in several standard rodent malaria models. In the Thompson test model (25) with Plasmodium berghei–infected ICR mice (fig. S3), ELQ-300 and P4Q391 were highly potent, with 50% effective doses (ED50) of 0.016 and 0.27 mg/kg per day, respectively (table S4). Cures were produced with doses as low as 0.1 mg/kg per day, and the lowest-dose group that resulted in no recrudescence within 30 days received 1.0 mg/kg per day. The therapeutic efficacy of ELQ-300 against P. yoelii and its pharmacokinetic parameters after oral administration in CD1 Swiss mice were assessed. The antimalarial activity of ELQ-300 was measured in direct Fig. 2. Ex vivo activity of ELQ-300 and P4Q-391 against Plasmodium clinical field isolates. (A) Ex vivo sus- comparison to atovaquone using a standard ceptibility of different Plasmodium species to ELQ-300 and P4Q-391 compared with that for other antima- Peters 4-day test, and blood parasitemia larials such as chloroquine. (B) Ex vivo susceptibility (median IC50 values) of P. falciparum (closed circles) was quantified by fluorescence-activated and P. vivax (open circles) to ELQ-300 and P4Q-391 compared to other antimalarials. P values were cal- cell sorting (FACS) analysis. ELQ-300 culated with Wilcoxon rank sum test. proved highly efficacious against P. yoelii; the dose that suppressed parasite prolifpressure for 8 weeks. Atovaquone-resistant parasites were observed on eration by 90% compared to vehicle (PEG400)–treated mice (ED90) day 30 in each of three atovaquone treatment flasks. In contrast, no was 0.15 mg/kg per day (Fig. 5). By comparison, the ED90 value for parasites could be detected after 8 weeks of treatment in the flasks atovaquone was 0.04 mg/kg per day. The drug exposure (AUC0–24 h) containing ELQ-300. Under the same culture conditions, seeding of of ELQ-300 in peripheral blood of infected mice after a single oral 10 parasites with no drug treatment resulted in detectable parasites administration at the ED90 was 1.4 mg hour/ml. Whereas atovaquone’s in 14 to 15 days. Thus, the lack of resistant parasites emerging within ED50 and ED90 values were superior, ELQ-300 was more powerful as a the 8-week period predicts a resistance frequency of 10,000

0.56

>18,000

P4Q-391

>10,000

1.0

>10,000

460

2.0

230


Atovaquone


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Fig. 4. EC50 curves for ELQ-300 and P4Q-391 against P. falciparum cytochrome bc1. Cytochrome c reductase activity was monitored by spectroscopic analysis with a dual-wavelength spectrophotometer in dual mode (550–541 nm). The assay was performed at 35°C in a stirred cuvette with 2,3-dimethoxy-5-methyl-6-decyl-1,4 benzohydroquinone (decylubiquinol) and horse heart cytochrome c in a buffered solution. Reactions were initiated by addition of hemozoin-free mitochondrial preparations.

Fig. 5. ELQ-300 activity compared with atovaquone activity against P. yoelii murine malaria. (A) Dose response for ELQ-300 versus atovaquone. Groups of n = 4 mice per group were infected with 6.4 × 106 P. yoelii–infected erythrocytes. Drug treatment was started 1 hour after infection and was administered on days 1 to 4. (B) Whole blood and plasma concentrations of ELQ-300 (nM) after oral gavage administration (target dose = 0.1 mg/kg) to P. yoelii–infected CD1 mice (n = 4 for each time point). Blood samples were taken after the last dose (day 4).

at day 7 after infection with respect to vehicle-treated mice was 5.9 mg/kg per day (Fig. 6). The estimated exposure of ELQ-300 at the ED90 dose in the SCID mouse model was AUCED90 = 1.1 mg hour/ml per day, which corresponds very closely to the potency observed in vivo against P. yoelii (AUCED90 = 1.4 mg hour/ml per day and ED90 = 0.15 mg/kg per day) in the 4-day test described above. Collectively, our in vivo data show that ELQ-300 and P4Q-391 are highly active against two different species of murine malaria and that ELQ-300 is equally potent against the human parasite P. falciparum in SCID mice. ELQ-300 and P4Q-391 were assessed for activity against exoerythrocytic stages of P. berghei in vitro. In this model, sporozoites of luciferaseexpressing P. berghei were added to a monolayer of HEPG2 hepatoma

cells and the infection was allowed to proceed for an hour. After this period, the drugs were diluted across the plate and the cultures were incubated at 37°C. Luciferase expression was determined at 44 hours after infection and the data were fit to a nonlinear logistic dose-response function. ELQ-300 and P4Q-391 were highly efficacious in this model with EC50 values of 1.02 and 5.11 nM, respectively (table S5). Next, we were interested in evaluating the quinolone-3-diphenylethers against Plasmodium cynomolgi because this nonhuman primate malaria species resembles the human malaria parasite P. vivax in that the liver stage involves both replicating and nondividing forms. ELQ-300 and P4Q-391 were tested in an in vitro liver-stage assay involving the infection of primary rhesus hepatocytes with sporozoites from the M strain of P. cynomolgi essentially as published (28), except for a 6-day exposure period. Compounds were evaluated in duplicate in a six-point threefold dilution series from 0.04 to 10 mM (table S6 and fig. S4). The parasiticidal effects were assessed by high-content fluorescence imaging to quantify the number of developing liver schizonts (large forms) and the small uninucleate parasite forms believed to be latent hypnozoites (28). After a 6-day incubation, both compounds showed remarkable activity against P. cynomolgi liver schizonts (IC50 ≈0.1 mM for both compounds, compared to 0.04 mM for primaquine, the positive control). Assessing the effect of the compounds on the small hypnozoite-like forms is complex because the number of small forms increases markedly in the presence of both of the quinolones. We interpret these results as indicative that schizont development is blocked at an early stage so the parasites remain small (or are killed and disappear from the culture). This same phenomenon has been observed for atovaquone and other antirespiratory compounds (29). An in vivo evaluation in the gold standard P. cynomolgi–infected rhesus monkey relapse model (30) will be used to address this issue in the future. The in vitro activity against liver stages of malaria prompted further evaluation in vivo with P. berghei (luciferase) sporozoite infections of BALB/c mice. In this model, five mice per group were infected and then dosed with ELQ-300 or P4Q-391 by the oral route 1 hour after infection. Efficacy was determined by bioluminescence imaging: at 44 hours and days 6 and 13 after infection. At each interval, mice were injected with D-luciferin, anesthetized, and imaged to monitor progression of liver- and blood-stage parasitemia. As shown in Fig. 7, both ELQ-300 and P4Q-391 were highly effective. No luminescence was observed in the liver at doses as low as 0.03 mg/kg and subsequent follow-up confirmed that the infections were completely blocked by single doses of ELQ-300 (≥0.03 mg/kg) and P4Q-391 (≥0.1 mg/kg). These data highlight the potential of 3-diarylether-4(1H)-quinolones for single-dose prophylaxis and potential cure. Effects of ELQ-300 and P4Q-391 on blocking transmission to the mosquito vector Antimalarials that prevent the transmission of malaria to the mosquito vector have an important role to play in malaria control programs and may help to contain the spread of drug resistance (31). To explore the potential transmission-blocking activity of the 4(1H)-quinolone-3diarylether chemotype, we investigated its ability to prevent development of stage V gametocytes, to kill late-stage gametocytes, and to prevent the development of viable ookinetes and oocysts in vitro and in vivo. Inhibition of gametocyte development by quinolone-3-diarylethers was evaluated in vitro. P. falciparum (NF54) gametocyte cultures were initiated using standard methods, and early-stage gametocytes (stages


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Fig. 6. Efficacy of ELQ-300 against the human malaria parasite in the immune-deficient (SCID) humanized mouse model of P. falciparum. A group of 3 mice treated with vehicle and another group of 11 mice treated with different doses of ELQ-300 were analyzed to estimate ED90 and AUCED90 as parameters of efficacy. (A) Parasitemia in individual mice treated with different doses of ELQ-300 during the efficacy assay. Each symbol represents an individual mouse. (B) Concentrations of ELQ-300 in the blood of each individual mouse in the efficacy study measured for 24 hours after the initial oral dose administration. Each symbol represents an individual mouse. (C) Graphic estimation of AUCED90 for ELQ-300. Data are the area under the curve (AUC) for ELQ-300 in blood during the first 24 hours after the first oral dose administration (AUC0→23 h) versus log10 (parasitemia at day 7) for each individual mouse in the efficacy study. (D) Microscopic and flow cytometry analysis of P. falciparum present in peripheral blood of mice treated with vehicle or ELQ-300. Samples were taken 48 hours after the start of treatment (that is, one cycle of exposure to drug). Flow cytometry dot plots from samples of peripheral blood show P. falciparum–infected human erythrocytes (circles). Images in the right-hand panels show Giemsa-stained blood parasites present at the 48-hour time point. Blood films from control untreated animals show normal staining and appearance, whereas the parasites in ELQ-300–treated animals stain poorly and exhibit altered morphology.

I to II) were exposed to 0.1, 1, and 10 mM ELQ-300 and P4Q-391 for 3 days. Drug pressure was then removed, and further development was assessed by microscopic examination of Giemsa-stained blood smears on days 3 and 7 after initiation of drug treatment (day 0). As shown in fig. S5, both drugs inhibited development of early-stage gametocytes. ELQ-300–treated gametocytes did not develop past stage III, even at 0.1 mM, the lowest dose tested. P4Q-391 was slightly less potent, with 1 mM exposure being the lowest dose to prevent development to stage IV. Development to stage IV was observed with exposure to 10 mM P4Q-391, which we attribute to the limited solubility of this compound. We also studied the effect of ELQ-300 on late-stage gametocyte viability. High-content fluorescence-based image analysis was used to measure the activity of ELQ-300 against late stage IV to V P. falciparum gametocytes (4, 32). The presence of ELQ-300 inhibited stage IV to V

Fig. 7. Whole-animal bioluminescence imaging of mice infected with luciferase-transfected P. berghei sporozoites. Mice were treated with different doses of ELQ-300, P4Q-391, or atovaquone. Animals (n = 5 per group) received a single dose by gavage 1 hour after inoculation with sporozoites. Representative images taken at 44 hours after infection are shown. Bioluminescent signal was detected in control untreated animals, with the highest intensity noted in the area overlaying the liver, consistent with the presence of liver-stage parasites. Notice that whereas all three drugs exhibited a dose-dependent effect on liver stages, ELQ-300 fully protected mice against P. berghei liver-stage infection at doses as low as 0.03 mg/kg.

gametocyte viability throughout a 72-hour period, with an IC50 value of 71.9 nM (fig. S6). Although the use of MitoTracker Red for viability assessment may be confounded by the mechanism of action of ELQ300, the level of activity observed was comparable to the internal control drug for this assay, puromycin (IC50 = 75.3 nM), and indicates a high level of activity against late-stage gametocytes. Both compounds were also evaluated in an in vitro P. berghei ookinete assay, which focuses on the first 24 hours of parasite development in the mosquito from gamete production, through fertilization and zygote formation, and, finally, to the development of the mature ookinete. Production of ookinetes was reported by expression of green fluorescent protein (GFP) controlled by an ookinete-specific promoter using a fluorescence endpoint (33, 34). ELQ-300 demonstrated high potency with an IC50 of 56 nM, similar to the atovaquone control (67 nM) and P4Q-391 (43 nM), providing further evidence of the transmissionblocking potential of these molecules. We evaluated the effect of ELQ-300 on in vivo P. berghei oocyst formation in Anopheles stephensi mosquitoes (35). Briefly, mouse blood infected with GFP-expressing P. berghei parasites was mixed with ELQ-300 and immediately placed into an array of membrane feeders at 39°C and offered to ≈75 overnight-starved mosquitoes. After 7 days, fluorescent oocysts were counted on the dissected midguts using a semiautomated image capture/analysis procedure with dimethyl sulfoxide (DMSO) as a negative control (36). In three separate experi-


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ments, the presence of ELQ-300 in the infectious blood feed had a marked impact on the mean oocyst numbers per mosquito. In one exemplary experiment (fig. S7), the prevalence of oocyst infections in the control group was 88.3%, which ranged from 0 to 496 oocysts per mosquito, yielding a geometric mean of 188.8 oocysts per mosquito. ELQ-300 demonstrated potent activity, that is, no oocysts formed in mosquitoes fed with infected blood containing 1 mM drug, and there was a 99.4% reduction of the total oocyst count relative to controls with blood containing 100 nM ELQ-300. The lowest concentration tested, 10 nM, reduced oocyst burden in mosquito midguts by 19.2%. Next, we added ELQ-300 to the blood meal of mosquitoes in the standard membrane-feeding assay (SMFA) with infected blood containing gametocytes of P. falciparum strain 3D7. The results from two independent experiments are presented in fig. S8. In one of the studies, ELQ-300 completely blocked P. falciparum oocyst formation in bloodfed mosquitoes at concentrations as low as 10 nM, whereas in the other study, a 95.9% reduction in oocyst formation was observed. These findings indicate that ELQ-300 is more effective against P. falciparum transmission stages than in the P. berghei rodent model. It is worth noting that the antimalarial spiroindolone NITD609 required concentrations of 500 nM to completely inhibit oocyst formation in a comparable SMFA (37). Together, these results demonstrate that ELQ-300 has the potential to affect malaria transmission by reducing the prevalence of infected mosquitoes and intensity of infection. The promising transmission-blocking activities of ELQ-300 and P4Q-391 in vitro prompted further evaluation in an in vivo model (fig. S9). In these studies, mice with about 3% parasitemia (P. berghei) were treated with a single oral dose of ELQ-300 and P4Q-391 or vehicle control at doses ranging from 0.1 to 10 mg/kg. Transmissionblocking efficacy was then determined by feeding female A. stephensi mosquitoes on the P. berghei–infected mice 1 hour after treatment. On day 10 after infection, the mosquitoes were dissected and midguts were examined for the presence of oocysts. Remarkably, doses of ELQ-300 and P4Q-391 as low as 0.1 mg/kg completely prevented transmission as measured by the development of oocysts. Positive controls for this experiment included dihydroartemisinin (10 mg/kg), which inhibited oocyst production by 63.7%, and primaquine (10 mg/kg), which inhibited oocyst development by 95.4%. Together, these data from in vitro and in vivo models demonstrate that ELQ-300 and P4Q-391 are promising drugs for malaria elimination and control efforts. Physiochemical and pharmacokinetic properties of ELQ-300 and P4Q-391 The lead optimization testing strategy included an assessment of the physicochemical, metabolic, and pharmacokinetic properties to identify features that would limit oral bioavailability and result in rapid clearance and poor exposure profiles. Early in the program, it was recognized that one of the most significant limitations of the series was very poor aqueous solubility, most likely due to the highly crystalline properties of these molecules. Unfortunately, all structural modifications that resulted in improved solubility also led to an unacceptable loss of in vivo efficacy. Solubility measurements (see the Supplementary Materials) were conducted using two different methods: a kinetic method that was more reflective of the procedures used in many of the in vitro test assays (that is, dilution of a DMSO stock solution, albeit into differing media) and a thermodynamic equilibrium method un-

der physiologically relevant conditions (that is, with respect to media and incubation time). Both methods suggested poor aqueous solubility (table S7); however, solubility results with the kinetic method were 10- to 50-fold higher than the thermodynamic measurements, which is consistent with the high crystallinity being a significant barrier to aqueous solubility. Given these results, particular attention was paid to all of the in vitro measurements to ensure that the compound was in solution under the conditions used in the individual assays. Although ELQ-300 and P4Q-391 both have low aqueous solubility, the remaining physicochemical properties (molecular weight, polar surface area, log P, and H-bond donors/acceptors) suggested that membrane permeability would likely not limit oral absorption and could help to attenuate the effect of poor solubility (table S7). The compounds also were found to be highly stable to metabolic degradation by cytochrome P450 enzymes during in vitro incubation with liver microsomes from multiple species, and they did not inhibit the major cytochrome P450 enzymes at concentrations needed for efficacy (table S8). After intravenous administration to mice, both compounds were found to have low clearance (~0.7 ml/min per kilogram) and moderate volumes of distribution (~1.2 liters/kg), with half-lives exceeding 15 to 20 hours (Fig. 8). These parameters are most likely influenced by the high plasma protein binding as shown in table S7. At an efficacious dose of 0.3 mg/kg with the PEG400 vehicle used in the P. yoelii efficacy experiments, the bioavailability of both compounds in mice was good (~100%) after oral administration. Under these dosing conditions, plasma concentrations were maintained at concentrations well above the in vitro P. falciparum EC50 for more than 48 hours after dosing. As the dose in mice was increased, the bioavailability decreased in line with solubilitylimited absorption. Studies in rats resulted in similar pharmacokinetic properties, although even at low doses, the oral bioavailability in rats was lower than that at low doses in mice (fig. S10 and table S9). Formulation approaches are currently in progress to address the solubility limitations and the lack of dose proportionality. Selectivity and safety profile of ELQ-300 and P4Q-391 On the basis of its superior intrinsic antiplasmodial activity in vitro and in vivo against blood and liver stages of malarial parasites, outstanding transmission-blocking activity, target selectivity, low propensity for resistance, ease of synthesis, and lack of cross-resistance against multidrug-resistant strains, ELQ-300 was chosen as the preclinical candidate from an impressive series of new 3-diarylether substituted 4(1H)-quinolones. To support further development of the drug, we performed additional safety profiling of ELQ-300. It is known that several antimalarial drugs, including halofantrine, block the hERG (human ether-a-go-go–related gene) channel and produce a QT interval prolongation, which is potentially life-threatening. hERG patch clamp assays with ELQ-300 yielded IC50 values of >11 mM, consistent with a low risk of cardiotoxicity (table S10). We also established that ELQ300 lacks mutagenic potential in a five-strain Ames bacterial assay, and the compound was negative for genotoxicity in an in vitro micronucleus assay. Finally, we did not observe any significant inhibition in a screen of 100 neurotransmitter receptors, peptide receptors, ion channels, reuptake sites, and enzymes of therapeutically relevant targets (table S11). On the basis of these studies, it appears that ELQ-300 has no significant off-target pharmacological activities that are a concern for human safety. Formulation studies are currently in progress to support the assessment of the safety margin in preclinical animal species.


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Fig. 8. ELQ-300 (filled circles) and P4Q-391 (open triangles) were administered intravenously and orally to nonfasted male Swiss outbred mice. (A and B) The intravenous dose (0.1 mg/kg) was formulated in mouse plasma to facilitate solubilization and to avoid precipitation upon administration via the tail vein. The oral dose was 0.3 mg/kg and was administered via gavage (0.1 ml) as a solution in undiluted PEG400. After intravenous administration (A), both of the drugs exhibited low clearance and a low

volume of distribution, with a long half-life of about 15 to 18 hours. The low clearance is consistent with in vitro data showing their high metabolic stability in the presence of hepatic microsomes (see the Supplementary Materials). After oral administration, the terminal half-life of each compound was similar to that after intravenous dosing, and absorption appeared to be slow with Tmax values of about 7.5 hours. The oral bioavailability of both ELQ-300 and P4Q-391 at 0.3 mg/kg was about 100%.

DISCUSSION

plete inhibition of P. berghei oocyst formation in a mouse-feeding study, thus totally inhibiting sporogony and demonstrating a 100% block of transmission. Unlike the structurally related pyridone GW844520 (12) from GSK and 4(1H)-quinolones recently reported by Biagini et al. (29), ELQ-300 has high in vitro selectivity for P. falciparum bc1 complex over the human enzyme complex and various mammalian cell types including hematopoietic stem cells. It does not inhibit a large panel of receptors and enzymes, including the hERG channel, nor is the compound genotoxic. In line with its low predicted dose in humans and its expected long half-life, ELQ-300 offers the potential of being used in combination with other antimalarial drugs to achieve the goal of a single-dose cure. Furthermore, the effects on liver and mosquito stages of the malaria life cycle offer the hope of a new molecule not only to help protect and cure patients but also to block transmission of malaria parasites to the mosquito vector, thus breaking the parasite life cycle. An exploratory formulation investigation is under way to support preclinical safety and toxicity studies before initiation of human clinical trials.

Quinolone-3-diarylethers represent a new antimalarial series active on a clinically validated pathway that overcomes the shortcomings of atovaquone, a clinical drug with the same enzyme target. From this potent series, ELQ-300 was selected as a preclinical candidate. ELQ300 is potent in vitro against blood-stage P. falciparum strains including a clinical isolate with a mutation located at the Qo binding region of cytochrome b that is associated with atovaquone resistance. Although additional biochemical studies are required to elucidate the antimalarial mechanism, it is interesting to speculate that ELQ-300 may target the Qi site of the enzyme complex. Doggett et al. (38) recently reported that a mutation in the Qi site region of the yeast cytochrome b gene greatly diminished the growth-inhibitory activity of ELQ-271. Although it rapidly inhibits oxygen respiration, the killing profile of ELQ-300 resembles atovaquone—with rapid killing coming only after an initial cytostatic phase. Repeated attempts to generate resistant mutants using single-step methodology have failed; thus, ELQ-300 has a demonstrated resistance frequency far improved over that for atovaquone. The in vitro potency, combined with high metabolic stability, results in excellent oral efficacy with a curative blood-stage dose of 1 mg/kg in the Thompson or Peters tests and activity in the P. falciparum SCID humanized mouse model that is consistent with these values based on blood exposure to the drug. The molecule is also highly potent against exo-erythrocytic stages and not only affects liver schizonts and gametocytes but also inhibits the formation of ookinetes and oocysts in the mosquito midgut. Although poorly soluble in aqueous media, ELQ300 exhibits good oral bioavailability at therapeutically relevant doses with extended half-lives in rodents. Impressively, in vivo doses lower than the ED90 result in causal prophylaxis and killing of all P. berghei primary liver schizonts; furthermore, such a dose also results in com-

MATERIALS AND METHODS In vitro antimalarial activity Laboratory strains of P. falciparum were cultured in human erythrocytes by standard methods under a low oxygen atmosphere (5% O2, 5% CO2, 90% N2) in an environmental chamber (39). The culture medium was RPMI 1640 supplemented with 25 mM Hepes buffer, gentamicin sulfate (25 mg/liter), hypoxanthine (45 mg/liter), 10 mM glucose, 2 mM glutamine, and 0.5% Albumax II (complete medium). The parasites were maintained in fresh human erythrocytes suspended at 2% hematocrit in complete medium at 37°C. Stock cultures were subpassaged every 3


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to 4 days by transfer of infected red cells to a flask containing complete medium and uninfected erythrocytes. SYBR Green assay. In vitro antimalarial activity of the 4(1H)quinolone-3-diarylether derivatives was assessed by the SYBR Green I fluorescence-based method (the “MSF assay”) described previously by us (15). Experiments were set up in triplicate in 96-well plates (Costar, Corning) with twofold dilutions of each drug across the plate in a total volume of 100 ml and at a final red blood cell concentration of 2% (v/v). The dilution series was initiated at a concentration of 1 mM, and the experiment was repeated beginning with a lower initial concentration for those compounds in which the IC50 value was below 10 nM. Automated pipetting and dilution was carried out with the aid of a programmable Precision 2000 robotic station (Bio-Tek). An initial parasitemia of 0.2% was attained by addition of normal uninfected red cells to a stock culture of asynchronous parasite-infected red cells. The plates were incubated for 72 hours at 37°C in an atmosphere of 5% CO2, 5% O2, and 90% N2. After this period, the SYBR Green I dye–detergent mixture (100 ml) was added and the plates were incubated at room temperature for 1 hour in the dark and then placed in a 96-well fluorescence plate reader (SpectraMax Gemini-EM, Molecular Diagnostics) for analysis with excitation and emission wavelength bands centered at 497 and 520 nm, respectively. The fluorescence readings were plotted against the logarithm of the drug concentration, and curve fitting by nonlinear regression analysis (GraphPad Prism software) yielded the drug concentration that produced 50% of the observed decline relative to the maximum readings in drug-free control wells (IC50). [3H]Hypoxanthine incorporation assay. The methods used were essentially as described by Desjardins et al. (16); however, the procedure was modified to a 72-hour incubation period. Ex vivo (P. falciparum and P. vivax field isolates) Compounds. Standard antimalarial drugs chloroquine, amodiaquine (Sigma-Aldrich), piperaquine, mefloquine, and artesunate (WWARN QA/QC Reference Material Programme) and experimental compounds ELQ-300 (Veterans Affairs Medical Center, Portland) and P4Q-391 (University of South Florida) were prepared as stock solutions (1 mg/ml) in DMSO. Drug plates were predosed by diluting the compounds in 50% methanol followed by lyophilization and stored at 4°C. Field location and sample collection. Plasmodium isolates were collected from patients attending malaria clinics in Timika (Papua, Indonesia), a region endemic for multidrug-resistant strains of P. vivax and P. falciparum. Patients with symptomatic malaria presenting to an outpatient facility were recruited into the study if singly infected with P. falciparum or P. vivax, with a parasitemia of between 2,000 and 80,000/ml, and most (>60%) of the parasites at the ring stage of development. Venous blood (5 ml) was collected by venipuncture, and after removal of host white blood cells with CF 11 cellulose, packed infected red blood cells were used for the ex vivo drug susceptibility assay. Ex vivo drug susceptibility assay. Drug susceptibility of P. vivax and P. falciparum isolates was measured with a protocol modified from the WHO microtest as described previously (20, 21, 40). Two hundred microliters of a 2% hematocrit blood media mixture, consisting of RPMI 1640 medium plus 10% AB+ human serum (P. falciparum) or McCoy’s 5A medium plus 20% AB+ human serum (P. vivax), was added to each well of predosed drug plates containing 11 serial concentrations (twofold dilutions) of the antimalarials (maximum concen-

tration shown in brackets): chloroquine (2991 nM), amodiaquine (81 nM), piperaquine (792 nM), mefloquine (338 nM), artesunate (25 nM), ELQ-300 (66 nM), and P4Q-391 (1042 nM). A candle jar was used to mature the parasites at 37°C for 30 to 56 hours. Incubation was stopped when >40% of ring-stage parasites had reached mature schizont stage in the drug-free control well. Thick blood films made from each well were stained with 5% Giemsa solution for 30 min and examined microscopically. The number of schizonts per 200 asexual-stage parasites was determined for each drug concentration and normalized to the control well. The dose-response data were analyzed with nonlinear regression analysis (WinNonLin 4.1, Pharsight Corporation), and the IC50 value was derived with an inhibitory sigmoid Emax model. Ex vivo IC50 data were only used from predicted curves, where Emax and E0 were within 15% of 100 and 1, respectively. Quality control procedures. Drug plate quality was assured by running schizont maturation assays (two independent experiments) with the chloroquine-resistant strain K1 and the chloroquine-sensitive strain FC27. For microscopy slide reading quality control, two randomly selected drugs per isolate were read by a second microscopist. If the raw data derived by the two microscopists lead to a marked shift in the IC50 estimates of the selected drugs, the whole assay (that is, all standard drugs and experimental compounds) was reread by a second reader and the results were compared. If necessary, discrepant results were resolved by a third reading by an expert microscopist. Fixed-ratio isobologram analysis of drug interactions between ELQ-300 and proguanil In vitro interaction of ELQ-300 and proguanil was assessed by isobolar analysis with fixed-ratio combinations, where drugs were diluted in fixed ratios of starting concentrations predetermined to generate well-defined concentration response curves (24). After determination of the intrinsic IC50 values for selected drugs, stock solutions were prepared with each drug at concentrations such that the final concentration in our 96-well drug susceptibility assay after four or five twofold dilutions approximated the IC50. If we call these stock solutions drug A and drug B, then six final stock solutions were prepared from this initial stock: drug A alone, drug B alone, and volume-volume mixtures of drugs A and B in the following ratios: 4:1, 3:2, 2:3, and 1:4. Twofold dilutions of each of the six final stock solutions were performed across a 96-well plate in quadruplicate. Initial data analysis yielded the intrinsic dose-response curve for each drug alone and four different fixed-ratio combination dose-response curves, with corresponding IC50 values. The fractional inhibitory concentration (FIC) was then calculated by the following formula: FIC (A) = IC50 of drug A in combination/IC50 of drug A alone; FIC (B) = IC50 of drug B in combination/IC50 of drug B alone; FIC index = FIC (A) + FIC (B). The isobolograms were constructed by plotting a pair of FICs for the combination of ELQ-300 and proguanil. An interpretation of a straight diagonal line (FIC index = 1) on the isobologram indicates an additive effect between the two drugs. A concave curve below the line (FIC index 1) indicates antagonism. Parasite reduction ratio The methods used for investigation of ELQ-300 parasiticidal kinetics, determination of PRR, and the time required to kill 99.9% of parasites in vitro, that is, parasite clearance time, 99.9%, have been described previously by Sanz et al. (23).


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In vivo therapeutic efficacy against P. yoelii (GSK) The in vivo efficacy of ELQ-300 against P. yoelii was evaluated in a standard 4-day test with minor modifications (41). Age-matched female CD1 Swiss (Harlan) mice were infected intravenously with 6.4 × 106 P. yoelii 17XNL–infected erythrocytes and randomly distributed in groups of n = 4. A range of doses of ELQ-300 solubilized in PEG400 were administered to mice once daily from 1 hour after infection (day 0) to day 3 by oral gavage. Parasitemia was measured in 2 ml of blood samples taken at day 4 of the assay using flow cytometry with the nucleic acid dye YOYO-1 as described (27). In vitro liver-stage activity (P. berghei) All mice used in these experiments were female BALB/c mice (average weight was about 18 g) obtained from Harlan. The transgenic P. berghei line 1052 cl1 (Pb1052 cl1) that expresses luciferase was used in the liver-stage studies and was obtained from C. J. Janse at Leiden University. HepG2 cells (75,000 per well) were seeded into collagencoated white 96-well plates for analysis with the TopCount microplate luminometer (Packard) as previously described (42, 43). Cells were maintained at 37°C in 5% CO2 in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 1.0% penicillin-streptomycin (Sigma), and 1.0% L-glutamine. Mosquito salivary glands were dissected, and 5000 sporozoites were added per well. Plates were incubated at 37°C for 3 hours and then washed three times with phosphate-buffered saline (PBS). Serial dilutions of experimental compounds were prepared, added to parasite-infected HepG2 cells in triplicate, and incubated at 37°C for 44 hours (44). After the incubation, cells were washed once with PBS and then lysed with 10 ml of cell culture lysis reagent (Promega Luciferase Assay System Kit). Immediately after cell lysis, 100 ml of luciferase assay substrate was added and then the parasite lysates were analyzed. Assessment of liver-stage development in vivo In vivo imaging of liver-stage parasites was performed as previously described (42, 43). Briefly, mice (n = 5 per group) were infected intravenously in the tail vein with P. berghei sporozoites (10,000 sporozoites per animal) and then dosed 1 hour after infection. Forty-four hours after infection, animals were injected intraperitoneally with D-luciferin (100 mg/kg), anesthetized with isoflurane, and imaged for liver-stage development. The IVIS Spectrum system was used to capture luciferase activity, and the Living Image 3.0 software was used to analyze the images (Caliper Life Sciences). This study was conducted in compliance with the Guide for the Care and Use of Laboratory Animals of the National Research Council for the National Academies. The protocol was approved by the University of South Florida Institutional Animal Care and Use Committee.

SUPPLEMENTARY MATERIALS www.sciencetranslationalmedicine.org/cgi/content/full/5/177/177ra37/DC1 Materials and Methods Fig. S1. PRR of ELQ-300. Fig. S2. Isobolograms of the interactions of ELQ-300 with proguanil against four different strains of P. falciparum (D6, Dd2, TM90-C2B, and V1/S). Fig. S3. Schematic overview of the Thompson test method for evaluation of candidate antimalarial drugs in mice. Fig. S4. Effect of drugs on P. cynomolgi large and small hepatic forms in vitro. Fig. S5. In vitro activity of ELQ-300 and P4Q-391 against development of early-stage gametocytes (P. falciparum).

Fig. S6. Comparative inhibitory activity of ELQ-300 and puromycin (control drug) on the development of stage IV to V P. falciparum in vitro. Fig. S7. Effect of ELQ-300 on the formation of oocysts in mosquitoes after feeding on a blood meal containing various concentrations of the drug. Fig. S8. Effect of ELQ-300 on oocyst development in blood-fed mosquitoes as assessed in a SMFA. Fig. S9. Schematic overview and results of in vivo transmission-blocking activity of ELQ-300 and P4Q-391 in mice infected with P. berghei. Fig. S10. Plasma concentration versus time profiles for ELQ-300 and P4Q-391. Table S1. Cytotoxicity evaluation of ELQ-300 against mammalian cells in culture. Table S2. Inhibition of human cytochrome bc1 by selected 4(1H)-quinolones. Table S3. Comparison of the propensity for resistance in P. falciparum (Dd2 strain) to ELQ-300, P4Q-391, and atovaquone. Table S4. In vivo efficacy of ELQ-300 and P4Q-391 in three different murine models of malaria infection. Table S5. In vitro liver-stage activity: P. berghei in HepG2. Table S6. IC50 values of the compounds tested against P. cynomolgi developing liver stages (large forms) and P. cynomolgi small forms (presumably hypnozoites). Table S7. Physicochemical properties and binding characteristics for ELQ-300 and P4Q-391. Table S8. Cytochrome P450 inhibition by ELQ-300 and P4Q-391. Table S9. Pharmacokinetic properties of ELQ-300 and P4Q-391 in mice and rats. Table S10. Summary of the blocking actions of ELQ-300 on hERG, KV1.5, NaV1.5, and CaV1.2 channels. Table S11. Inhibition by ELQ-300 of selected off-target receptors, ion channels, and biogenic amine transporters (% of no-inhibitor control values at 10 mM ELQ-300).

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RESEARCH ARTICLE and R.E.S. evaluated ELQ-300 in ookinete, oocyst, and SMFAs; A.N.L., F.E.S., and D.E.K. carried out in vivo transmission-blocking assays including bioluminescence imaging; P.S. provided guidance and oversight on issues relating to in vitro safety assessment; A.N., A.N.L., S.A.C., A.B.V., D.E.K., J.N.B., R.M., and M.K.R. wrote the paper; M.K.R., R.M., S.A.C., A.B.V., D.E.K., R.W.W., R.K.G., and J.N.B. provided overall management and oversight of the project and collaboratively designed the studies, analyzed the data, and led the candidate selection process; M.K.R. and R.M. were project leaders for their respective teams, whereas J.N.B. and I.B. (deceased) served as overall project directors. Competing interests: R.K.G. is a member of the Medicines for Malaria Venture Expert Scientific Advisory Committee and is compensated for this activity. He has served as the Chair of that Committee. I.A.-B., M.B.J.-D., S.F., E.H., L.M.S., and F.-J.G. are employees of GSK, and the experimental work by them has been carried out in the GSK Diseases of the Developing World in Tres Cantos, Spain. M.K.R., J.X.K., R.W.W., A.N., J.N.B., D.E.K., R.M., and R.M.C. are named as co-inventors on the following patent applications related to this work: WO/2010/065905 and WO/2012/167237. The other authors declare that they have no competing interests. Submitted 25 September 2012 Accepted 28 February 2013 Published 20 March 2013 10.1126/scitranslmed.3005029 Citation: A. Nilsen, A. N. LaCrue, K. L. White, I. P. Forquer, R. M. Cross, J. Marfurt, M. W. Mather, M. J. Delves, D. M. Shackleford, F. E. Saenz, J. M. Morrisey, J. Steuten, T. Mutka, Y. Li, G. Wirjanata, E. Ryan, S. Duffy, J. X. Kelly, B. F. Sebayang, A.-M. Zeeman, R. Noviyanti, R. E. Sinden, C. H. M. Kocken, R. N. Price, V. M. Avery, I. Angulo-Barturen, M. B. Jiménez-Díaz, S. Ferrer, E. Herreros, L. M. Sanz, F.-J. Gamo, I. Bathurst, J. N. Burrows, P. Siegl, R. K. Guy, R. W. Winter, A. B. Vaidya, S. A. Charman, D. E. Kyle, R. Manetsch, M. K. Riscoe, Quinolone-3diarylethers: A new class of antimalarial drug. Sci. Transl. Med. 5, 177ra37 (2013).


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in isolation of parasite mitochondria. We are also grateful to M. Santos Martínez (Drug Metabolism and Pharmacokinetics); M. J. Almela, S. Lozano (Parasitology and Cell Biology), and the Therapeutic Efficacy, Pharmacology and Laboratory Animal Science groups at GSK Tres Cantos Medicines Development Campus for assistance. We thank L. D. Shultz and The Jackson Laboratory for providing access to nonobese diabetic scid IL2Rgc null mice through their collaboration with GSK Tres Cantos Medicines Development Campus. We wish to dedicate this work to the memory of Dr. Ian Bathurst (1949–2011). Funding: Supported by the Medicines for Malaria Venture and by NIH grants AI28398 (M.W.M. and A.B.V.), R01 GM097118-01 (R.M. and D.E.K.), and AI079182 and AI100569 (M.K.R.). M.K.R. also receives funding from the U.S. Department of Veterans Affairs and the Stanley Medical Research Institute. C.H.M.K. receives additional support from the Wellcome Trust. Author contributions: A.N., R.W.W., D.E.K., R.M.C., R.M., J.N.B., and M.K.R. contributed to drug design; A.N. and R.W.W. synthesized ELQ-300; R.M.C. and R.M. synthesized P4Q-391; T.M., D.E.K., Y.L., and J.X.K. conducted in vitro susceptibility against laboratory-adapted strains and cytotoxicity assays; J.M., G.W., and B.F.S. conducted the in vitro studies of field isolates, with program supervision from R.N.P. and R.N.; Y.L. and J.X.K. performed in vitro drug synergy studies; L.M.S. and F.-J.G. determined the PRR for ELQ-300; A.-M.Z. and C.H.M.K. evaluated drugs against P. cynomolgi; E.H. conducted in vitro ATP depletion assays; A.N.L., F.E.S., and D.E.K. evaluated drugs for therapeutic efficacy against blood-stage infections with P. berghei in mice; I.A.-B. and M.B.J.-D. evaluated drugs for in vivo efficacy against P. falciparum and P. yoelii blood-stage infections in mice and S.F. determined the pharmacokinetics of each drug in these efficacy experiments; Y.L. and J.X.K. assessed drugs for in vivo efficacy against blood-stage infections (P. yoelii) in mice; A.N.L., T.M., and D.E.K. conducted in vitro and in vivo liver-stage determinations; J.M.M. and A.B.V. conducted propensity for resistance experiments; I.P.F., M.W.M., A.B.V., and M.K.R. conducted enzyme inhibitor assays; K.L.W., D.M.S., J.S., E.R., and S.A.C. determined the pharmacokinetics of each drug in this investigation in uninfected preclinical animals species; A.N.L., F.E.S., and D.E.K. evaluated drugs for inhibition of gametocyte development (in vitro); S.D. and V.M.A. conducted late-stage gametocyte viability assays; M.J.D.