A novel multiple-stage antimalarial agent that inhibits protein synthesis

Abstract

There is an urgent need for new drugs to treat malaria, with broad therapeutic potential and novel modes of action, to widen the scope of treatment and to overcome emerging drug resistance. Here we describe the discovery of DDD107498, a compound with a potent and novel spectrum of antimalarial activity against multiple life-cycle stages of the Plasmodium parasite, with good pharmacokinetic properties and an acceptable safety profile. DDD107498 demonstrates potential to address a variety of clinical needs, including single-dose treatment, transmission blocking and chemoprotection. DDD107498 was developed from a screening programme against blood-stage malaria parasites; its molecular target has been identified as translation elongation factor 2 (eEF2), which is responsible for the GTP-dependent translocation of the ribosome along messenger RNA, and is essential for protein synthesis. This discovery of eEF2 as a viable antimalarial drug target opens up new possibilities for drug discovery.

Extended Data Figure 1. Synthetic Methodology

a. Synthesis of DDD107498 (4). b. Synthesis of DDD102542 (6a) and DDD103679 (6b).

Extended Data Figure 2. In vitro Activity

a. In vitro activity against a panel of resistant and sensitive strains of P. falciparum. Abbreviations: chloroquine (CQ), pyrimethamine (PYR), cycloguanil (CYC), quinine (QUI), sulfadoxine (SUL) and/or mefloquine (MFQ). Resistance as follows: K1 (CQ, SUL, PYR, CYC); W2 (CQ, SUL, PYR, CYC); 7G8 (CQ, PYR, CYC); TM90C2A (CQ, PYR, MFQ, CYC); D6 (MFQ); V1/S (CQ, SUL, PYR, CYC). Data are the means ± s.d. of n=3 independent [³H]hypoxanthine incorporation experiments (each run in duplicate). b. Ex-vivo activity against P. falciparum and P. vivax clinical isolates from Papua (Indonesia). c. Effect of DDD107498 on P. falciparum 3D7, HepG2 and MRC5 cells. Data are the means ± s.d. of n reported independent experiments.

Extended Data Figure 3. Effect of DDD107498 on parasite morphology

a. Phenotype of P. falciparum in peripheral blood of NOD-scid IL-2R_null mice engrafted with human erythrocytes. Blood samples were taken at day 5 and 7 of the assay (1 and 2 asexual cycles, respectively) after the start of treatment with vehicle or DDD107498 at day 3. The bidimensional flow cytometry plots measure the murine (Ter-119-PE+) and human (Ter-119-PE−) erythrocytes, and the presence of nucleic acids (infected SYTO-16+ events). The blue circles indicate the region of infected erythrocytes. Vehicle-treated mice showed a characteristic pattern of staining with SYTO-16, which correlated with the presence of healthy rings, trophozoites and schizonts in blood smears. Conversely, mice treated with DDD107498 at 50 mg/kg showed only trophozoites with condensed cytoplasm and some pyknotic cells at day 5 (red circle in flow cytometry plot and corresponding blood smears). By day 7, few infected erythrocytes were detected by flow cytometry and blood smears revealed parasites with a similar morphology to those at day 5. This suggests that trophozoites are the most sensitive population since the cycle is interrupted at this stage. The images displayed are taken from a mouse with high levels of parasitemia. At least 50 parasites were counted per sample screened in the microscope. Of these, 4 photos of representative parasite phenotype were selected to represent the morphology of the most prevalent phenotype. Thus, this is a qualitative assessment. b. Stage specificity assays using synchronised cultures. For morphological analysis of antimalarial drug action, thin blood smears were prepared, fixed and stained with Giemsa followed by examination with an upright microscope using an oil-immersion lens (100×). For parasitemia determination, a total number of 1000 red blood cells (corresponding to 5 microscopic fields) were counted. R to T. Abnormal trophozoites observed after 24h exposure of synchronized rings to DDD107498. T to S. Trophozoites do not develop into schizonts after 24h exposure to DDD107498. S-R. No ring stages are observed 24h after treatment of schizonts with DDD107498. c. Percentage parasitemia in the red blood cells. R = ring stage, T = trophozoite, S = schizont.

Extended Data Figure 4. Prophylactic activity of DDD107498 against sporozoite challenge

P. berghei (luciferase) sporozoite in vivo mouse model of chemoprotection. A dose of 3mg/kg was fully protective. Data are the mean of n=5.

Extended Data Figure 5. DDD107498 in vitro activity on the different life cycle stages of Plasmodium spp.

For comparator data with known antimalarials see Figure 5 in Delves et al. DDD107498 also showed potent activity in vitro against P. berghei ookinetes, (5.0nM [CI 4.4-5.7nM]), confirming that if DDD107498 was taken up during a blood meal, it could continue to kill parasites within the mosquito.

Extended Data Figure 6. In vivo P. berghei mouse-to-mouse assay

a. Impact of DDD107498 treatment on in vivo mosquito infection. Mice infected with P. berghei (PbGFPCON507 clone 1) were dosed orally with either DDD107489 at 3 mg/kg, atovaquone at 0.3 mg/kg, sulphadiazine at 8.4 mg/kg, or were not drug treated (negative control). After 24 h, populations of treated mice (n=5) were exposed to 500 female A. stephensi mosquitoes, and oocyst intensity and infection prevalence in the mosquito midgut was measured 10 days post-feeding. Individual data points represent the number of oocysts found in individual mosquitoes. Two replicates were performed. Sulphadiazine (8.4 mg/kg, i.p.) and atovaquone (0.3 mg/kg, i.p.) were used as negative and positive transmission-blocking drug controls, respectively. Horizontal bars indicate mean intensity of infection, whilst error bars indicate S.E.M. within individual mosquito populations. b. Impact of treatment with DDD107498 over a complete transmission cycle in vivo. Following drug treatment of infected mice and mosquito feeds, surviving potentially infectious mosquitoes were allowed to blood-feed on naive mice at a range of transmission settings (biting rates of 2,5 and 10 bites per naive mouse) to assess ability of drug treatment to reduce the number of new malarial cases post mosquito bite. Efficacy is expressed as: i) impact on the mosquito population – expressed as reduction in both oocyst and sporozoite intensity and prevalence; ii) impact on subsequent transmission to new naïve vertebrate hosts – expressed as reduction in infection of naïve mice (reduction in number of new malarial cases post-mosquito bite) and inhibition of subsequent parasitema (day 10 post-bite) in mice that do become infected; iii) Effect size generated – by fitting the data achieved within this assay to a chain-binomial model we can assess the ability of DDD107498 to reduce R_o (assuming 100% coverage). If R_o is reduced to <1, transmission is unsustainable and elimination will occur. 95% Cls are shown in brackets. Efficacy is calculated in comparison with no-drug controls. TB is transmission blocking

Extended Data Figure 7. ClustalWS alignment of eEF2 sequences from Human (Hs), Yeast (Sc) and Plasmodium falciparum (Pf)

Alignment made using Jalview ClustalX default colouring.

Extended Data Figure 8. Fitness phenotypes of DDD107498-resistant parasite lines

Unmarked Dd2 and DDD107498-selected parasites with various levels of resistance were assessed for growth in a competition assay, relative to a Dd2-GFP reference line. a. Equal numbers of unmarked test lines were mixed with the Dd2-GFP reference, in triplicate wells, and the ratio of non-fluorescent and fluorescent cells assessed by flow cytometry over time. At day 0, all lines had a 1:1 ratio with the Dd2-GFP reference. Increased growth of the test line over the Dd2-GFP reference, which has a slower growth rate than unmarked WT Dd2, would result in an increased ratio of Test:Dd2-GFP. b. Growth assay of 4 different test lines: i) WT Dd2, ii) EF2-E134D, iii) EF2-L775F, and iv) EF2-Y186N, relative to Dd2-GFP. A faster growth rate of WT Dd2 (DDD107498 IC₅₀ 0.14 nM) relative to the fluorescent Dd2-GFP line is reflected in an increased ratio over time. The low-level resistant line EF2-E134D (IC₅₀ 5.8 nM) did not attain a WT growth rate, and the high-level resistant lines EF2-L775F (IC₅₀ 660 nM) and EF2-Y186N (3100 nM) were further impaired. Means ± s.d.; n=4 independent experiments each run in triplicate.

Figure 1. Chemical evolution of DDD107498 from the phenotypic hit

Cli = intrinsic clearance in mouse liver microsomes.

Figure 2. Efficacy studies and parasite killing rate

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 < 2-3 %. The percentage of parasitemia was calculated by acquiring a minimum number of 500 parasitized erythrocytes. An independent experiment with 3 further mice was performed to confirm the ED₉₀. b. Determination of the in vitro killing rate of DDD107498. The in vitro PRR assay was used to determine onset of action and rate of killing as previously described. 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 described. Four independent serial dilutions were done with each sample to correct for experimental variation and the error bars shown are the standard deviation. Previous results reported on standard antimalarials tested at 10 × EC₅₀ using the same conditions are shown for comparison. c. The in vitro PRR 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 shown are the standard deviation. Concentrations of DDD107498 ≥ 1 × EC₅₀ are sufficient to produce maximal killing effects on treated parasites.

Figure 3. In vitro activity of DDD107498 against P. berghei liver stages

hpi is hours post-infection. Each time point was the average of 4 technical replicates. 95% Confidence limits are shown. * more than one curve fitting possible.

Figure 4. eFF2

a. eEF2 promotes the GTP-dependent translocation of the ribosome along mRNA during protein synthesis. b. Homology model of Plasmodium 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 >50 parasites visualized on two independent occasions. d. Protein and DNA/RNA synthesis were evaluated by measuring the incorporation of [³⁵S]-labelled methionine and cysteine ([³⁵S]-Met/Cys) (upper panel) and [³H]-labelled hypoxanthine (lower panel) into asynchronous 3D7 wild-type (○) and 3D7 DDD107498-resistant line (eEF2-E134A/P754A) (●) after 40 min incubation with DDD107498, cycloheximide or actinomycin D. Radiolabeled incorporation, measured as cpm, was normalised as % 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).