ddd107498  (Cyprotex Discovery)

Journal: Life Science Alliance

Article Title: Differential effects of translation inhibitors on Plasmodium berghei liver stage parasites

doi: 10.26508/lsa.202302540

Figure Lengend Snippet: (A) Mean fluorescence intensity (MFI) of the nascent proteome in experimentally matched liver stage parasite populations at 28 and 48 hpi after 4-h DMSO treatment, as detailed in schematics; n = 5 independent experiments, P = 0.001 (two-tailed, unpaired t test). SPZ, sporozoite infection denotes timepoint = 0; OPP, o-propargyl-puromycin labeling; ACFM, automated confocal feedback microscopy. (A, B) Single parasite OPP-MFI at 28 hpi after a 4-h acute pre-treatment (as in schematic in (A)) with 3.7 nM bruceantin or DMSO control. Boxplots show combined data from two independent experiments, with dotted lines reporting treatment means, whereas single points represent individual exoerythrocytic forms (EEFs), colored by experiment. (A, C) SuperPlots of single parasite OPP-MFI at 28 hpi after a 4-h acute pre-treatment (as in schematic in (A)) with DDD107498 or DMSO control. Single points show individual EEFs, colored by independent experiment; experiment means are represented by large circles, and bars represent the mean and SD. These raw data are extracted from the five-point dose–response dataset published in normalized form in of reference . (D) Representative confocal images of competitive LS translation inhibition by 100 μM LysRS-IN-2 versus DMSO control, as detailed in experiment schematics. Merged images are pseudocolored as indicated with EEFs immunolabeled with α- Pb HSP70, OPP-A555 labeling the nascent proteome, and DNA stained with Hoechst. Scale bars = 5 μm.

Article Snippet: Anisomycin (176880; EMD Millipore/Sigma-Aldrich), bruceantin (HY-N0840; MedChem Express), DDD107498 (A8711; Apex Bio), Lys-RS-IN-2 (Y-126130; MedChem Express), and MMV019266 (STK845176; Vitas-M Laboratory) were solubilized in DMSO (D2650; Sigma-Aldrich), aliquoted, and stored at −20°C.

Techniques: Fluorescence, Two Tailed Test, Infection, Labeling, Microscopy, Control, Inhibition, Immunolabeling, Staining

Journal: Life Science Alliance

Article Title: Differential effects of translation inhibitors on Plasmodium berghei liver stage parasites

doi: 10.26508/lsa.202302540

Figure Lengend Snippet: (A, B) Translation inhibition was quantified in P. berghei exoerythrocytic forms (A, B) and matching in-image HepG2 (A) after acute pre-treatment from 24 to 28 hpi, as shown in the schematic. Compounds were tested in an 8- or a 10-point, threefold serial dilution. (A) Concentration–response curves. Single data points represent the mean translation at each concentration, normalized to DMSO controls, and error bars show the SD from n = 3 independent experiments. The absence of line indicates that no curve was fit. (B) Single LS parasite concentration–response. For each compound, concentrations are plotted moving from lowest (left) to highest (right) as indicated by the triangles. The top concentrations tested were 3,333 nM for anisomycin and DDD107498, 123 nM for bruceantin, 16,667 nM for MMV019266, and 100,000 nM for LysRS-IN-2. N = 3 independent experiments combined; each dot represents the mean translation intensity (OPP-MFI) in a single parasite normalized to DMSO controls, n = 10,461 exoerythrocytic forms in total. The full concentration–response dataset can be explored via interactive dashboards in our KNIME hub workflow: https://hub.knime.com/-/spaces/-/∼TZCrKvv3sbJwM_xP/current-state/ . (C) Experimental setup to probe the effects of acute translation inhibition and the relationship between translation inhibition efficacy and antiplasmodial efficacy with inhibitors tested at equivalent effective concentrations. Source data are available for this figure.

Article Snippet: Anisomycin (176880; EMD Millipore/Sigma-Aldrich), bruceantin (HY-N0840; MedChem Express), DDD107498 (A8711; Apex Bio), Lys-RS-IN-2 (Y-126130; MedChem Express), and MMV019266 (STK845176; Vitas-M Laboratory) were solubilized in DMSO (D2650; Sigma-Aldrich), aliquoted, and stored at −20°C.

Techniques: Inhibition, Serial Dilution, Concentration Assay

Journal: Life Science Alliance

Article Title: Differential effects of translation inhibitors on Plasmodium berghei liver stage parasites

doi: 10.26508/lsa.202302540

Figure Lengend Snippet: Single parasite translation quantified at 48 hpi after 24 h of treatment with 20 nM DDD107498 and DMSO controls. Single data points are individual exoerythrocytic forms, normalized to DMSO controls from n = 2 independent experiments as color-coded in the legend. Boxplots show combined data from both experiments, with dotted lines reporting treatment means. P < 0.0001 in an unpaired, two-tailed t test.

Article Snippet: Anisomycin (176880; EMD Millipore/Sigma-Aldrich), bruceantin (HY-N0840; MedChem Express), DDD107498 (A8711; Apex Bio), Lys-RS-IN-2 (Y-126130; MedChem Express), and MMV019266 (STK845176; Vitas-M Laboratory) were solubilized in DMSO (D2650; Sigma-Aldrich), aliquoted, and stored at −20°C.

Techniques: Two Tailed Test

Journal: Life Science Alliance

Article Title: Differential effects of translation inhibitors on Plasmodium berghei liver stage parasites

doi: 10.26508/lsa.202302540

Figure Lengend Snippet: (A, B, C) Experimental schematic for panels (B, C) quantifying translation recovery and growth at 48 hpi after DDD107498 treatment of anisomycin-arrested parasites in early schizogony. (B) Representative single confocal images of translation in P. berghei LS at 48 hpi. Merged images are pseudocolored as indicated with parasite (exoerythrocytic form) immunolabeled with α-HSP70, OPP-A555 labeling the nascent proteome, and DNA stained with Hoechst. Scale bar = 5 μm. (C) Single parasite translation and size quantified at 48 hpi. Single points show individual exoerythrocytic forms color-coded by independent experiments, with experimental means represented by large circles (n = 2), and bars represent the mean and SD of the experiments. (D) Experimental schematic for quantifying merosome release in experiment-matched wells at 72, 96, and 120 hpi, after DDD107498 treatment of translationally arrested early LS schizonts and subsequent washout. Stacked bar charts (E, H) report the total merosomes collected per timepoint from all experiments. (F, I) Percentage of total detached cells/merosomes released after 72 hpi (% delayed) are reported as means with error bars showing the SD. (E, F, G, H, I, J) Total detached cell/merosome release normalized to the DMSO controls, with error bars showing the SD. Data are shown from n = 3 (E, F, G) or n = 2 (H, I, J) independent experiments; DDD498 = DDD107498 in the axis labels for (E, F, G, H, I, J).

Article Snippet: Anisomycin (176880; EMD Millipore/Sigma-Aldrich), bruceantin (HY-N0840; MedChem Express), DDD107498 (A8711; Apex Bio), Lys-RS-IN-2 (Y-126130; MedChem Express), and MMV019266 (STK845176; Vitas-M Laboratory) were solubilized in DMSO (D2650; Sigma-Aldrich), aliquoted, and stored at −20°C.

Techniques: Immunolabeling, Labeling, Staining

Journal: Scientific Reports

Article Title: Evaluating antimalarial efficacy by tracking glycolysis in Plasmodium falciparum using NMR spectroscopy

doi: 10.1038/s41598-018-36197-3

Figure Lengend Snippet: Evaluating the effect of various potent antimalarial compounds on glycolytic activity of ring and trophozoite stage P. falciparum . The plots depict the fold-change in glycolytic rate as measured for ring (R) and trophozoite (T) stage parasites treated with 1 µM each of atovaquone ( a ), cladosporin ( b ), and DDD107498 ( c ). In case of artemisinin, treatment was done for two different time periods of 5 h ( d ) and 10 h ( e ). The raw data for lactate C3 peak integral values, for ring and trophozoite stage parasites, from 2 h NMR measurements, from replicate experiments, is shown in Supplementary Fig. . All the experiments were performed in duplicates and the error bars represent standard deviation for drug vs control. ( f ) Giemsa stained smears showing the morphology of control and artemisinin treated (10 h) ring and trophozoite stage parasites, before (10 h) and after (12 h) NMR measurements.

Article Snippet: 10 mM stock solutions of the atovaquone, artemisinin, cladosporin, and 5.18 mM stock solution of DDD107498 were prepared in cell culture grade DMSO (Sigma-Aldrich, US) and stored in −20 °C until use.

Techniques: Activity Assay, Standard Deviation, Staining

Journal: Malaria Journal

Article Title: The Plasmodium falciparum cytoplasmic translation apparatus: a promising therapeutic target not yet exploited by clinically approved anti-malarials

doi: 10.1186/s12936-018-2616-7

Figure Lengend Snippet: Half-maximal effective concentrations (nM) determined in P. falciparum growth inhibition assay

Article Snippet: All other compounds were purchased from the indicated vendors: DDD107498 (Apexbio #A8711-5), mefloquine hydrochloride (Sigma-Aldrich #M2319), emetine (Sigma-Aldrich #E2375), cycloheximide (Fisher #AC35742-0010), MMV008270 (Vitas-M Laboratory #STK591252), actinomycin D (Sigma-Aldrich #A1410), tubercidin (Sigma-Aldrich #T0642), thapsigargin (Sigma-Aldrich #SML1845), ionomycin (Sigma-Aldrich #407951), thiostrepton (Sigma-Aldrich #598226), bruceantin (Toronto Research Chemicals #B689310), verrucarin A (Sigma-Aldrich #V4877), anisomycin (Sigma-Aldrich #A5862), homoharringtonine (Sigma-Aldrich #SML1091), lactimidomycin (EMD Millipore #506291), nagilactone C (BOC Sciences #24338-53-2), suramin sodium salt (Sigma-Aldrich #S2671), puromycin (Thermo Fisher #A1113803), halofuginone (Sigma-Aldrich #32481).

Techniques: Inhibition

Journal: Malaria Journal

Article Title: The Plasmodium falciparum cytoplasmic translation apparatus: a promising therapeutic target not yet exploited by clinically approved anti-malarials

doi: 10.1186/s12936-018-2616-7

Figure Lengend Snippet: Dose-response curves of Pf growth, PfIVT, and RRIVT for mefloquine and controls. Dose-response curves comparing inhibition, calculated as % inhibition, of P. falciparum growth assay (black), P. falciparum in vitro translation assay (red), and commercially available rabbit reticulocyte in vitro translation assay (purple) for a cycloheximide, b DDD107498 (M5717), and c mefloquine. Name of compound, mechanism of action (where definitively known), and molecular structure are displayed at top of each graph

Article Snippet: All other compounds were purchased from the indicated vendors: DDD107498 (Apexbio #A8711-5), mefloquine hydrochloride (Sigma-Aldrich #M2319), emetine (Sigma-Aldrich #E2375), cycloheximide (Fisher #AC35742-0010), MMV008270 (Vitas-M Laboratory #STK591252), actinomycin D (Sigma-Aldrich #A1410), tubercidin (Sigma-Aldrich #T0642), thapsigargin (Sigma-Aldrich #SML1845), ionomycin (Sigma-Aldrich #407951), thiostrepton (Sigma-Aldrich #598226), bruceantin (Toronto Research Chemicals #B689310), verrucarin A (Sigma-Aldrich #V4877), anisomycin (Sigma-Aldrich #A5862), homoharringtonine (Sigma-Aldrich #SML1091), lactimidomycin (EMD Millipore #506291), nagilactone C (BOC Sciences #24338-53-2), suramin sodium salt (Sigma-Aldrich #S2671), puromycin (Thermo Fisher #A1113803), halofuginone (Sigma-Aldrich #32481).

Techniques: Inhibition, Growth Assay, In Vitro