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The World Health Organization estimates that there were approximately 200 million clinical cases and 584,000 deaths from malaria in 2013, predominantly among children and pregnant women in sub-Saharan Africa1. The malaria parasite has developed resistance to many of the current drugs, including emerging resistance to the core artemisinin component of artemisinin-based combination therapies that constitute current first-line therapies2. To support the current treatment and eradication agenda3, there are several requirements for new antimalarials: novel modes of action with no cross-resistance to current drugs; single-dose cures; activity against both the asexual blood stages that cause disease and the gametocytes responsible for transmission; compounds that prevent infection (chemoprotective agents); and compounds that clear Plasmodium vivax hypnozoites from the liver (anti-relapse agents)4.

Discovery of a novel antimalarial

A phenotypic screen of the Dundee protein kinase scaffold library5 (then 4,731 compounds) was performed against the blood stage of the multi-drug-sensitive Plasmodium falciparum 3D7 strain. A compound series from this screen, based on a 2,6-disubstituted quinoline-4-carboxamide scaffold, had sub-micromolar potency against the parasites, but suffered from poor physicochemical properties. Chemical optimization (Fig. 1 and Extended Data Fig. 1) led to DDD107498 with improved physicochemical properties (Tables 1 and 2 in Supplementary Methods) and a 100-fold increase in potency. The key stages involved were replacing the bromine with a fluorine atom to reduce molecular mass and lipophilicity; replacing the 3-pyridyl substituent with an ethyl-pyrrolidine group; and addition of a morpholine group via a methylene spacer. Initial cost-of-goods estimates and likely human dose projections suggest a low cost (approximately US$1 per treatment), which is important, given that most of the patient population is living in poverty.

Figure 1: Chemical evolution of DDD107498 from the phenotypic hit.
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Calculated log(P), calculated log(partition coefficient); Solubility, solubility in water; Mouse cli., intrinsic clearance in mouse liver microsomes.

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Blood-stage activity and developability

DDD107498 showed excellent activity against 3D7 parasites: 50% effective inhibitory concentration (EC50) = 1.0 nM (95% confidence interval (CI) 0.8–1.2 nM); EC90 = 2.4 nM (95% CI 2.0–2.9 nM); EC99 = 5.9 nM (95% CI 4.5–7.6 nM), (n = 39). It was also almost equally active against several drug-resistant strains (Extended Data Fig. 2a)6. Furthermore, DDD107498 was more potent than artesunate in ex vivo assays against a range of clinical isolates of both P. falciparum (median EC50 = 0.81 nM (range 0.29–3.29 nM), n = 44) and P. vivax (median EC50 = 0.51 nM (range 0.25–1.39 nM), n = 28) collected from patients with malaria from southern Papua, Indonesia, a region where high-grade multidrug-resistant malaria is endemic for both species (Extended Data Fig. 2b)7,8. In contrast, the compound was not toxic to human cells (MRC5 and Hep-G2 cells) at much higher concentrations (>20,000-fold selectivity; Extended Data Fig. 2c).

DDD107498 showed good drug-like properties: metabolic stability when incubated with hepatic microsomes or hepatocytes from several species; good solubility in a range of different media; and low protein binding (Tables 1 and 2 in Supplementary Methods). DDD107498 displayed excellent pharmacokinetic properties in preclinical species, including good oral bioavailability (an important prerequisite for use in resource-poor settings) and long plasma half-life (important for single-dose treatment and chemoprotection) (Extended Data Table 1a).

DDD107498 was very active in several mouse models of malaria, with comparable or greater efficacy than current antimalarials (Extended Data Table 1b). DDD107498 had a 90% reduction in parasitaemia (ED90) of 0.57 mg per kg (body weight) after a single oral dose in mice infected with the rodent parasite Plasmodium berghei. Efficacy was also tested in NOD-scid IL-2R_null mice engrafted with human erythrocytes and infected with P. falciparum strain 3D70087/N9 (Fig. 2a)9. When orally dosed daily for 4 days, the ED90 on day 7 after infection was 0.95 mg per kg per day. Blood sampling from the infected SCID (severe combined immunodeficiency) mice suggested a minimum parasiticidal concentration for DDD107498 of 10–13 ng ml−1 for asexual blood-stage infections.

Figure 2: Efficacy studies and parasite killing rate.
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a, In vivo activity against P. falciparum in NOD-scid IL-2R_null mice. Six mice were used, each serially sampled. The variability of cytometry for repeated acquisitions of a sample is less than 2–3%. The percentage of parasitaemia was calculated by acquiring a minimum number of 500 parasitized erythrocytes. An independent experiment with three further mice was performed to confirm the ED90. b, Determination of the in vitro killing rate of DDD107498. The in vitro parasite reduction rate assay was used to determine onset of action and rate of killing as previously described10. P. falciparum was exposed to DDD107498 at a concentration corresponding to 10 × EC50. The number of viable parasites at each time point was determined as described10. Four independent serial dilutions were done with each sample to correct for experimental variation; error bars, s.d. Previous results reported on standard antimalarials tested at 10 × EC50 using the same conditions are shown for comparison10. c, The in vitro parasite reduction rate assay was used to determine the minimal concentration of compound needed for achieving maximal killing effects. Parasites were exposed to DDD107498 at concentrations of 0.1, 0.3, 1, 3 and 10 × EC50 using conditions described above. Error bars, s.d. Concentrations of DDD107498 of 1 × EC50 are sufficient to produce maximal killing effects on treated parasites.

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The effects of DDD107498 on circulating parasites in the SCID mouse model could be observed in one replication cycle (48 h) and led to trophozoites with condensed cytoplasm (Extended Data Fig. 3). Stage specificity studies using synchronized cultures showed that, at a concentration of 4 nM for 24 h, DDD107498 led to (1) from the ring stage, formation of abnormal trophozoites; (2) from the trophozoite stage, prevention of schizont formation with a 50% reduction in parasites, indicative of cidal activity; and (3) from the schizont stage, prevention of ring formation with a 98% reduction in parasites, indicative of cidal activity (Extended Data Fig. 3b, c).

DDD107498 showed a similar parasite killing profile both in vitro (Fig. 2b, c) and in vivo (Fig. 2a), which is supportive of a common mode of action in cellular and animal models of disease. Using a parasite reduction rate assay10 there was a lag of about 24–48 h, during which time the effects of the compounds were reversible following wash-out. Rapid killing occurred after parasites had been exposed to DDD107498 for more than 48 h (Fig. 2b, c).

All these experiments suggest for the blood-stage form that treatment with DDD107498 prevented development of trophozoites and schizonts and, at least in the case of schizonts, caused rapid killing. Any ring-stage parasites only developed as far as abnormal trophozoites, which appeared to survive for about 48 h under drug pressure, but were then killed.

In safety studies, DDD107498 showed no clinically relevant inhibition of any of the major human cytochrome P450 (CYP) isoforms and CYP450 induction risk was low (Table 3 in Supplementary Methods), indicating a low risk of clinical drug–drug interactions. DDD107498 is non-mutagenic and has very weak inhibitory potencies on IKr (hERG) and other ion channels, indicating a very low risk for adverse cardiovascular activity. Given its potency, long half-life observed in preclinical species, and safety margins from a rat 7-day toxicology study, DDD107498 has potential both for single-dose treatment and for once-weekly chemoprotection11.

Activity against other life-cycle stages

Intra-hepatocytic parasites (liver schizont stages) are the first stage of human infection after injection of sporozoites by anopheline mosquitoes. Compounds active against this stage have potential for use in chemoprotection. DDD107498 showed an EC50 ≈ 1 nM against the liver schizont forms of P. berghei and Plasmodium yoelii12. DDD107498 was active when dosed for only 2 h during the initial infection (hepatocyte invasion) of the liver cells (Fig. 3). In contrast, atovaquone (clinically used for chemoprotection in combination with proguanil) had a much reduced activity during this period (EC50 ≈ 106 nM versus 0.3 nM for continuous treatment). Further, DDD107498 showed equivalent potency against the P. berghei liver stage when added after initial infection had been established (Fig. 3). This suggests that intermittent treatment may be sufficient for chemoprotection. To assess chemoprotective potential in vivo, mice were treated with DDD107498 2 h before being infected with luciferase-expressing P. berghei sporozoites (Extended Data Fig. 4). At a dose of 3 mg per kg, DDD107498 was fully curative with no sign of parasitaemia after 30 days. Thus, DDD107498 demonstrates potent chemoprotection using in vitro and in vivo models, where blood sampling from the mice during the experiment suggests a minimum parasiticidal concentration of 15–20 ng ml−1.

Figure 3: In vitro activity of DDD107498 against P. berghei liver stages.
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Each experiment was the average of four technical replicates; h.p.i., hours post-infection. Bars, 95% CI. *More than one curve fitting possible.

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The parasite erythrocytic form differentiates into the asymptomatic male and female gametocytes (stages I–V) within the human host. Mature stage V gametocytes are infective to mosquitoes, but are not eliminated by the majority of current antimalarial agents, and remain circulating in the human bloodstream for up to 3 weeks, long after the disappearance of clinical symptoms of malaria13,14. After ingestion by the mosquito, gametocytes differentiate into gametes. DDD107498 potently inhibited both male and female gamete formation from the gametocyte stage at similar concentrations (1.8 nM (95% CI 1.6–2.1 nM) and 1.2 nM (95% CI 0.8–1.6 nM) respectively; Extended Data Fig. 5), indicating that it is an extremely potent inhibitor of the functional viability of both male and female mature gametocytes15. In line with this, DDD107498 blocked transmission, as determined by the standard membrane feeding assay. In this assay, parasite cultures containing P. falciparum stage V gametocytes were exposed to compound for 24 h before mosquito feeding. DDD107498 blocked subsequent oocyst development in the mosquito (measured after 7 days) with an EC50 of 1.8 nM. At a baseline oocyst intensity of 27 oocysts per mosquito in the DMSO controls, prevalence of infection was inhibited, with an EC50 of 3.7 nM, as measured by the number of infected mosquitoes. Repeating the standard membrane feeding assay in which DDD107498 was added at the moment of mosquito feeding gave an EC50 of 10 nM, indicating potent activity against the parasite sexual stages that develop in the mosquito midgut (Extended Data Fig. 5)16.

A P. berghei mouse-to-mouse17,18 model was additionally used to examine transmission blockade. Mice were infected with P. berghei (PbGFPCON507)19, and then treated orally with compound 24 h before mosquitoes took a direct blood meal17. After a 3 mg per kg dose of DDD107498, a 90.7% reduction in infected mosquitoes and a 98.8% reduction in oocysts per midgut was observed at day 10 compared with mosquitoes fed on untreated mice (Extended Data Fig. 6). A corresponding reduction in sporozoite intensity and prevalence was observed (93.8% and 88.6% respectively). Mosquitoes previously fed on infected, drug-treated mice were allowed to feed on uninfected mice17. Testing a range of mosquito biting rates, we observed a mean reduction of 89.5% (95% CI 71.4–100) in the number of mice that developed blood-stage infection compared with mice bitten by mosquitoes that had fed on non-drug-treated infected mice. The overall effectiveness of an intervention over a round of transmission (from mouse to mosquito to mouse) can be quantified by estimating its ability to reduce the basic reproductive number (R0). This has been termed the ‘effect size’ of an intervention. By fitting data from the mouse-to-mouse assay to a chain binomial model we can estimate the effect size of the intervention17, assessing the ability of DDD107498 usage to reduce the basic reproductive number R0 (assuming 100% coverage). Our results estimate an effect size of 90.5% (95% CI 78.3–94.2), suggesting that DDD107498 is capable of acting as a potent transmission-blocking drug over multiple transmission settings within a field context18. The combination of these key in vitro and in vivo assays demonstrates the very strong potential of DDD107498 for blocking transmission; importantly, the required doses are likely to be similar to those required for treatment of blood-stage malaria.

DDD107498 targets PfeEF2

To determine the molecular target for DDD107498, asexual blood-stage P. falciparum were cultured in the presence of DDD107498 at 5 × EC50, until parasites became resistant (Extended Data Table 2)20. Resistance was obtained in the 3D7 (drug-sensitive) and 7G8 and Dd2 (multi-drug-resistant) strains, with minimum inocula of 107, 107 and 106 parasites respectively. Genomic DNA was extracted from resistant lines and whole-genome sequencing of ten drug-resistant lines identified shared mutations in one gene, which were not present in the parental lines: Pf3D7_1451100 (Supplementary Data 1). This gene encodes P. falciparum translation elongation factor 2 (PfeEF2). Three lines had two single nucleotide polymorphisms in PfeEF2, with a mixture of wild-type (WT) and mutant reads at each position (Supplementary Data 2), suggesting that these lines were mixtures of two clones, each with independent mutations in PfeEF2. Single nucleotide polymorphisms were confirmed by Sanger sequencing and nine validated mutations in PfeEF2 clustered in three regions of the encoded protein. In two cases identical single nucleotide polymorphisms were identified in two independent lines, indicating mutations in functionally important residues that were acquired independently in separate selection experiments. The fact that resistance to DDD107498 can be associated with multiple independent mutations is in keeping with observations from both artemisinin and other antimalarial compounds in development21,22,23.

eEF2 is one of several essential elongation factors required in eukaryotic protein synthesis, by mediating GTP-dependent translocation of the ribosome along messenger RNA (mRNA) (Fig. 4a)24. Yeast eEF2 is the target of the antifungal compound sordarin25,26. Sordarin is selective for the Saccharomyces cerevisiae eEF2 over mammalian eEF2 in vitro, demonstrating that although protein synthesis in eukaryotes is conserved, it is possible to obtain selective inhibitors, despite the relatively high homology (67.2% identity) between the yeast and human eEF2 (Extended Data Fig. 7)25,27. In keeping with the potential for selectivity, DDD107498 is not toxic to mammalian cells (Extended Data Fig. 2c). The PfeEF2 mutations associated with resistance mapped to several areas on the surface of a homology model of the protein (on the basis of the structure of S. cerevisiae eEF2 with no ligands bound28), with the mutations giving the highest degree of resistance clustering together (Fig. 4b). A DDD107498 binding pocket could not be elucidated through these modelling studies.

Figure 4: DDD107498 targets protein synthesis via eEF2.
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a, eEF2 promotes the GTP-dependent translocation of the ribosome along messenger RNA during protein synthesis. b, Homology model of P. falciparum eEF2. The mapped mutations from each strain are colour coded by EC50 fold (red, high; amber, moderate; green, low). c, Live-cell imaging of P. falciparum expressing an extra copy of eEF2 (WT) fused to GFP. The image is representative of more than 50 parasites visualized on two independent occasions. d, Protein and DNA/RNA synthesis were evaluated by measuring the incorporation of [35S]Met/Cys (upper panel) and [3H]hypoxanthine (lower panel) into asynchronous 3D7 wild-type (open circles) and 3D7 DDD107498-resistant lines (eEF2-E134A/P754A) (filled circles) after incubation for 40 min with DDD107498, cycloheximide or actinomycin D. Radiolabelled incorporation, measured as counts per minute, was normalized as the percentage of incorporation against inhibitor concentration (means ± s.d.; n = 3 independent experiments, each run in duplicate). e, The EC50 values for transfectants against DDD107498 (means ± s.d.; n = 4–7 independent experiments, each run in duplicate). Statistical significance was determined by the Mann–Whitney U-test: *P < 0.05; **P < 0.01. f, DDD107498-resistant line (eEF2-Y186N) transfected episomally with plasmids expressing either WT eEF2 or eEF2-Y186N (means ± s.d.; n = 3 independent experiments, each run in duplicate).

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We confirmed that brief pre-incubation with DDD107498 specifically inhibited P. falciparum protein synthesis, by measuring short-term incorporation of [35S]-labelled methionine and cysteine ([35S]Met/Cys) in WT and DDD107498-resistant 3D7 P. falciparum parasites (Fig. 4d)21. Cycloheximide (a protein synthesis inhibitor) and DDD107498 both prevented [35S]Met/Cys incorporation into WT P. falciparum 3D7, whereas actinomycin D (an inhibitor of RNA transcription) had minimal effect. Notably, DDD107498 was 100-fold less effective at inhibiting protein synthesis in DDD107498-resistant 3D7 than WT parasites, whereas cycloheximide prevented [35S]Met/Cys incorporation equally in resistant and WT lines. DDD107498 and cycloheximide had minimal effects on DNA/RNA biosynthesis both in sensitive and in drug-resistant parasites (measured by incorporation of [3H]-labelled hypoxanthine), demonstrating specificity. In contrast, actinomycin D caused a marked dose-dependent reduction in [3H] incorporation.

To confirm that PfeEF2 is the target for DDD107498, we integrated transgenes expressing either WT PfeEF2 or resistance-associated alleles of PfeEF2 (Y186N, observed in mutant Dd2; and P754S, observed in mutant 3D7) using attPxattB integrase-mediated recombination21,29. These transfectants also express endogenous PfeEF2. Imaging of green fluorescent protein (GFP)-fusions of PfeEF2 showed cytoplasmic localization (Fig. 4c), indicating that DDD107498 inhibits protein synthesis in the cytoplasm as opposed to the apicoplast30, the site of action of tetracycline and azithromycin.

Dose–response assays with DDD107498 showed a similar inhibition profile between PfeEF2 transgene-expressing lines and the WT Dd2 strain, indicating that the endogenous WT PfeEF2 has a dominant effect in these experiments (Fig. 4e). This may be due to stable complex formation between the ribosome, WT PfeEF2 and DDD107498, resulting in ribosome stalling, which would explain why in mixed populations the WT PfeEF2 is dominant. For example in bacteria, fusidic acid binds to the complex between EF-G and the ribosome, preventing dissociation and blocking protein translation31.

To determine whether WT PfeEF2 was dominant in poisoning translation, we introduced episomal plasmids encoding WT or Y186N mutant PfeEF2 into the resistant PfeEF2 Y186N line (Fig. 4f). Plasmid-borne PfeEF2-Y186N had no effect on sensitivity to DDD107498 (EC50 ≈ 3,100 nM), whereas WT PfeEF2 restored sensitivity (EC50 = 2 nM). This demonstrated a dominant effect of WT PfeEF2 on parasite susceptibility and confirmed that PfeEF2 is the primary molecular target of the compound. We note that the shallow slope observed for the WT PfeEF2 transfected line is probably a result of heterogeneous episomal plasmid copy number seen with episomally transformed parasite lines29. Structural studies will be required to define precisely how DDD107498 interacts with eEF2 and the ribosome.

Resistance has been reported for all clinical antimalarials, including artemisinins2. While the correlation between the rate of resistance generation in laboratory and clinical settings for antimalarials is not fully understood, it is important to evaluate the risk of all new antimalarials both in preclinical and in clinical studies. In our studies, the minimum inoculum for generating resistance to DDD107498 is within acceptable limits32. Furthermore, selected resistant Dd2 lines revealed impaired growth rates in the absence of drug pressure compared with WT Dd2 (Extended Data Fig. 8); moreover the higher the resistance, the greater the fitness defect. Importantly genome sequences from 1,685 clinical isolates of P. falciparum from 17 countries23,33 reveal a high degree of PfeEF2 sequence conservation in the field. The sole non-synonymous single nucleotide polymorphism (T16S) identified was unique to West Africa (allele frequency of 0.002) and is in a PfeEF2 domain distinct from mutations associated with in vitro resistance to DDD107498.

Conclusion

DDD107498 represents a promising prospect for development as an antimalarial agent, with a potent activity profile against multiple life-cycle stages (sub-10 nM), a novel mode of action and excellent drug-like properties. It has potential for single-dose treatment, which has major implications for ensuring patient compliance and practical deployment. Its complementary activity on the sexual stages of the parasite has potential to reduce transmission and its action on the liver stage suggests a possible role in chemoprotection. Chemoprotection and transmission-blocking properties are fundamental to the goal of eliminating and eradicating malaria, for which the high potency and long half-life of DDD107498 are well suited.

Owing to general concerns about the emergence of resistance, all antimalarials are developed as combination therapies, a strategy shown to improve efficacy and reduce the development of drug resistance. In terms of treatment of the erythrocytic stage, DDD107498 fulfils the criteria as a long-duration partner to complete the clearance of blood-stage parasites11. Therefore, it should be combined with a fast-acting compound, ideally with a pharmacological duration of action as close to DDD107498 as possible. This would reduce the initial level of infection, with the prolonged activity of DDD107498 eliminating the remaining parasites11.

Inhibition of protein synthesis by DDD107498 through PfeEF2, which is expressed in multiple life-cycle stages34, provides mechanistic support for the observed broad-spectrum profile. This highlights PfeEF2 as a novel drug target in malaria, and implies that inhibition of protein synthesis is an effective intervention for achieving multi-stage activity in Plasmodium. DDD107498 has now been progressed into advanced non-clinical development, with the aim of entering into human clinical trials.