A drug repurposing approach reveals targetable epigenetic pathways in Plasmodium vivax hypnozoites
- Center for Tropical and Emerging Global Disease, University of Georgia, United States
- Calibr, a division of The Scripps Research Institute, United States
- Malaria Molecular Epidemiology Unit, Institute Pasteur of Cambodia, Cambodia
- Novartis Institute for Tropical Diseases, Novartis Institutes for Biomedical Research, United States
- Shoklo Malaria Research Unit, Mahidol-Oxford Tropical Medicine Research Unit, Thailand
- Department of Molecular, Cell, and Systems Biology, University of California, Riverside, United States
- Center for Vaccines and Immunology, College of Veterinary Medicine, University of Georgia, United States
- International Center for Malaria Research, Education and Development, Emory Vaccine Center, Emory National Primate Research Center, Emory University, United States
- Medicines for Malaria Venture (MMV), Switzerland
- BioIVT Inc, United States
- Division of Infectious Diseases, Department of Medicine, Emory University, United States
- Department of Computer Science and Engineering, University of California, Riverside, United States
- Department of Microbiology and Immunology, University of Otago, New Zealand
- School of Sciences, Clayton State University, United States
- Mahidol Vivax Research Unit, Mahidol University, Thailand
- Department of Entomology, Armed Forces Research Institute of Medical Sciences (AFRIMS), Thailand
- Department of Chemistry, University of California, Riverside, United States
- Environmental Toxicology Graduate Program, University of California, Riverside, United States
- Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, United Kingdom
Figures
Figure 1 with 3 supplements
Hypnozonticidal hit detection and confirmation.
(A) Index chart depicting the primary screen of the ReFRAME library against P. vivax hypnozoites in an 8-day assay. Hypnozoite counts were normalized by mean quantity per well for each plate (Z-score). Teal: library, black: DMSO, red: 1 μM monensin. (B) Dose–response curves for cadralazine against P. vivax and P. cynomolgi liver forms in 8-day assays at the IPC, UGA, and NITD. All replicate wells were plotted together from all independent experiments (n = 3 for P. vivax at IPC, n = 1 for P. vivax at NITD, n = 2 for P. cynomolgi at UGA, and n = 4 for P. cynomolgi at NITD), bars represent SEM.
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Figure 1—source data 1
Source data for Figure 1 and supporting figures.
- https://cdn.elifesciences.org/articles/98221/elife-98221-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
ReFRAME screen run detail and hit structures.
(A) Index chart from Figure 1A with phosphatidylinositol 4-kinase inhibitor (PI4Ki) KDU691 or MMV390048, tafenoquine, and atovaquone controls added. Teal circle: library, black square: DMSO, pink triangle: 1 μM monensin, light green inverted triangle: 1 μM P4Ki, black diamond: 1 μM atovaquone, purple square: 10 μM tafenoquine. Some hits discussed in this report are noted with black circles; P: poziotinib, B: budralazine, H: hydralazine, C: cadralazine. (B) Simple linear regression correlating Z-factor with average hypnozoite count per well. (C) Structures of hits which confirmed to be active against P. vivax hypnozoites in dose–response assays; blue: hydralazine analogs, purple: other novel hits, green: re-discovery of compounds previously demonstrated to have hypnozonticidal activity in vitro or antirelapse activity in vivo.
Figure 1—figure supplement 2
Select ReFRAME hits confirmed at Novartis Institute for Tropical Diseases (NITD).
Dose–response curves for hydralazine and poziotinib against P. vivax liver forms assayed at NITD. All replicate wells were plotted together from a single independent experiment, bars represent SEM.
Figure 1—figure supplement 3
Pharmacokinetics of cadralazine in nonhuman primates.
Mean plasma concentration of cadralazine was measured in three male rhesus macaques after oral dosing. Plasma was collected following a 1 mg/kg dose, and again following a 30 mg/kg dose. Bars represent SD. The approximate IC50 and IC90 from P. vivax hypnozoite assays are indicated.
Figure 2 with 1 supplement
Synergistic effect of cadralazine and 5-azacytidine in P. vivax liver stage assays.
(A) Isobologram of cadralazine and 5-azacytidine activity against hypnozoites in fixed ratios of 1:0, 8:1, 6:1, 4:1, 2:1, 1:1, 1:2, 1:4, 1:6, 1:8, and 0:1, bars represent SD of FICs from two independent experiments. (B) Dose–response curves for cadralazine at the most synergistic fixed ratios (2:1, 4:1, and 8:1) against hypnozoites. Cadralazine alone is represented as 1:0, 5-azacytidine alone is represented as 0:1 and plotted on the cadralazine chart for comparison. Left and right charts represent two independent experiments, bars represent replicate wells at each dose.
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Figure 2—source data 1
Source data for Figure 2 and supporting figures.
- https://cdn.elifesciences.org/articles/98221/elife-98221-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Synergistic effect of cadralazine and 5-azacytidine in P. vivax liver stage assays.
Dose–response curves for cadralazine with all fixed ratios of 5-azacytidine against P. vivax hypnozoites. Cadralazine alone is represented as 1:0, 5-azacytidine alone is represented as 0:1 and plotted on the cadralazine chart for comparison. Left and right charts represent two independent experiments.
Figure 3 with 5 supplements
Cytosine modifications in P. vivax liver forms.
(A) Immunofluorescent imaging of a 5mC-positive (left) or 5hmC-negative (right) P. vivax hypnozoite (top) and schizont (bottom) at day 6 post-infection. White arrows indicate hepatocyte nuclei positive for 5mC or 5hmC. Bars represent 10 µm. (B) High-content quantification of 5mC or 5hmC stain area within hypnozoites or schizonts from sporozoites generated from three different P. vivax cases. Significance determined using Kruskal–Wallis tests, for hypnozoites H(7) = 194.3, p < 0.0001, for schizonts H(7) = 88.66, p < 0.0001, with Dunn’s multiple comparisons, *p < 0.05, ***p < 0.001, ****p < 0.0001, ns = not significant. Line, box, and whiskers represent median, upper and lower quartiles, and minimum-to-maximum values, respectively, of all hypnozoites (177 ≤ n ≤ 257) or all schizonts (30 ≤ n ≤ 142) in culture for each case, 2’ indicates a secondary stain only control.
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Figure 3—source data 1
Source data for Figure 3 and supporting figures.
- https://cdn.elifesciences.org/articles/98221/elife-98221-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Cytosine modifications in P. vivax liver forms, full panels from case 1 (expanded from Figure 3).
(A) Immunofluorescent imaging of a 5mC-positive P. vivax hypnozoite (top) and schizont (bottom) at day 6 post-infection. (B) Immunofluorescent imaging of a 5hmC-negative P. vivax hypnozoite (top) and schizont (bottom) at day 7 post-infection. Yellow arrows indicate autofluorescence in the blue channel associated with cell debris above the hepatocyte monolayer. White arrows indicate hepatocyte nuclei which are dimly stained with Hoechst 33342 and positive for 5mC or 5hmC. Purple arrows indicate 5mC-positive foci within the parasite. Bars represent 20 µm.
Figure 3—figure supplement 2
Cytosine modifications in P. vivax liver forms, full panels from case 2.
(A) Immunofluorescent imaging of a 5mC-positive P. vivax hypnozoite (top) and schizont (bottom) at day 6 post-infection. (B) Immunofluorescent imaging of a 5hmC-negative P. vivax hypnozoite (top) and schizont (bottom) at day 7 post-infection. Yellow arrows indicate autofluorescence in the blue channel associated with cell debris above the hepatocyte monolayer. White arrows indicate hepatocyte nuclei which are dimly stained with Hoechst 33342 and positive for 5mC or 5hmC. Purple arrows indicate 5mC-positive foci within the parasite. Bars represent 20 µm.
Figure 3—figure supplement 3
Cytosine modifications in P. vivax liver forms, full panels from case 3.
(A) Immunofluorescent imaging of a 5mC-positive P. vivax hypnozoite (top) and schizont (bottom) at day 6 post-infection. (B) Immunofluorescent imaging of a 5hmC-negative P. vivax hypnozoite (top) and schizont (bottom) at day 7 post-infection. Yellow arrows indicate autofluorescence in the blue channel associated with cell debris above the hepatocyte monolayer. White arrows indicate hepatocyte nuclei which are dimly stained with Hoechst 33342 and positive for 5mC or 5hmC. Purple arrows indicate 5mC-positive foci within the parasite. Bars represent 20 µm.
Figure 3—figure supplement 4
High-content analysis of cytosine modifications and P. vivax liver stage population metrics.
(A) Masks used to quantify parasite area and 5mC or 5hmC signal, (i) raw image taken with a 20x objective, (ii) Mask for P. vivax liver stages, (iii) mask for 5mC or 5hmC signal, and (iv) intersection of parasite mask (light blue) and 5mC or 5hmC signal mask (yellow), leading to quantified area of signal per form. (B) Histogram of growth area all parasites quantified for Cases 1, 2, and 3 in Figure 3. Hypnozoites were classified as forms with an area of 125 µm2 and smaller.
Figure 3—figure supplement 5
Cytosine modifications in P. cynomolgi M/B strain liver forms.
(A) Immunofluorescent imaging of a 5mC-positive P. cynomolgi hypnozoite (top) and schizont (bottom) at day 8 post-infection. (B) Immunofluorescent imaging of a 5hmC-negative P. cynomolgi hypnozoite (top) and schizont (bottom) at day 8 post-infection. Yellow arrows indicate autofluorescence in the blue channel associated with cell debris above the hepatocyte monolayer. White arrows indicate hepatocyte nuclei which are dimly stained with Hoechst 33342 and positive for 5mC or 5hmC. Purple arrows indicate 5mC-positive foci within the parasite. Bars represent 20 µm. (C) High-content quantification of 5mC or 5hmC stain area within hypnozoites or schizonts. Experiment 1 was fixed at day 8 post-infection, Experiment 2 was fixed at day 12 post-infection. Significance was determined using Kruskal–Wallis tests for hypnozoites and schizonts, with Dunn’s multiple comparisons, ****p < 0.0001, ns = not significant. Line, box, and whiskers represent median, upper and lower quartiles, and minimum-to-maximum values, respectively, of all hypnozoites (124 ≤ n ≤ 712) or all schizonts (7 ≤ n ≤ 581) in culture, 2’ indicates a secondary stain only control. Images in A, B are from Experiment 1.
Figure 4 with 3 supplements
Density of cytosine and methylated cytosine (5mC) in sporozoites.
(A) CG content of chromosome 1 for P. vivax and P. cynomolgi. The total number of cytosines was quantified on each strand using 1 kbp long non-overlapping windows. (B) The total number of methylated cytosines was quantified on each strand using 1 kbp long non-overlapping windows. (C) The number of 5mC present in all possible contexts (CG, CHG, and CHH) quantified throughout the genome of P. vivax and P. cynomolgi. (D) Repartitioned 5mC quantity within different compartments of the genome in P. vivax and P. cynomolgi. (E) Strand specificity of 5mC for all genes in P. vivax and P. cynomolgi. Flanking regions and gene bodies were divided into five bins, and the methylation level of each bin was averaged among all genes. Red: template strand, blue: non-template strand. (F) The previously reported mRNA abundance of P. vivax sporozoites was retrieved (Antonova-Koch et al., 2018) and genes ranked. The 5mC levels in 5′ flanking regions, gene bodies, and 3′ flanking regions were placed into five bins and are shown for highly expressed (90th percentile, left) and weakly expressed (10th percentile, right) genes. Red: template strand, blue: non-template strand.
Figure 4—figure supplement 1
Measurement of DNA methylation and DNA methyltransferase (DNMT) in P. vivax and P. cynomolgi sporozoites.
(A) Liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis of 5mC or 5hmC from enzymatically digested gDNA from P. vivax sporozoites, P. cynomolgi sporozoites, and P. falciparum blood stage parasites, as well as negative controls including uninfected mosquito salivary glands and ovaries from the same colony of mosquitoes used to generate the respective sporozoites. Bars represent SD of two independent experiments. (B) DNMT activity measured from nuclear extracts of P. vivax sporozoites, P. cynomolgi sporozoites, and uninfected mosquito salivary glands using the Epiquick DNMT activity assay. Data are from a single experiment.
Figure 4—figure supplement 2
Cytosine and methylation density plots for P. vivax sporozoites.
(A) CG content of chromosome 1–14 (Chr 1–14). The total number of cytosines quantified on each strand using 1 kb long non-overlapping windows. (B) The total number of methylated cytosines quantified on each strand using 1 kb long non-overlapping windows.
Figure 4—figure supplement 3
Cytosine and methylation density plots for P. cynomolgi sporozoites.
(A) CG content of chromosome 1–14 (Chr 1–14). The total number of cytosines quantified on each strand using 1 kb long non-overlapping windows. (B) The total number of methylated cytosines quantified on each strand using 1 kb long non-overlapping windows.
Figure 5 with 3 supplements
Characterization of primary human hepatocyte (PHH) metabolism following 1-aminobenzotriazole (1-ABT) treatment.
PHH lot BGW was seeded in 384-well plates and cultured for 7 days before treatment with 100 μM 1-ABT for 1 hr, followed by addition of substrates for 1 hr and collection for analysis by mass spectrometry. Data are combined from two independent experiments, bars represent SD of all replicates. Significance determined by Student’s t tests, ****p < 0.0001, ***p < 0.001, **p < 0.01, ns, not significant.
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Figure 5—source data 1
Source data for Figure 5 and supporting figures.
- https://cdn.elifesciences.org/articles/98221/elife-98221-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Monensin activity in all control wells based on primary human hepatocyte (PHH) lot.
(A) Initially, the ReFRAME was screened with cryopreserved vials of a specific lot of PHH, UBV. Screening continued with a new lot, BGW, once the supply of UBV vials was exhausted. The activity of monensin was significantly reduced in wells with BGW versus UBV PHH. A Mann–Whitney test indicates the difference was statistically significant, U(NUBV = 105, NBGW = 41) = 552, z = –2.15, ****p < 0.0001. (B) Metabolic activity panel for PHH lots UBV and BGW performed as part of regular quality control at the vendor (BioIVT). ECOD: 7-ethoxycoumarin O-deethylation, UGT: 7-hydroxycoumarin glucuronidation, ST: 7-hydroxycoumarin sulfation, CYP 1A2: phenacetin O-deethylation, CYP 2A6: coumarin 7-hydroxylation, CYP 2B6: bupropion hydroxylation, CYP 2C8: amodiaquine N-desethylation, CYP 2C9: tolbutamide methyl-hydroxylation, CYP 2C19: S-mephenytoin 4′-hydroxylation, CYP 2D6: dextromethorphan O-demethylation, CYP 2E1: chlorzoxazone 6-hydroxylation, CYP 3A4 (T): testosterone 6β-hydroxylation, CYP 3A4 (M): midazolam 1-hydroxylation. (C) PHH lot BGW was seeded into 384-well plates and cultured for 7 days before addition of a dilution series of 1-aminobenzotriazole (1-ABT) in media. Cytochrome P450 3A4 activity (CYP3A4) was measured using luciferin-IPA (Promega). RLU: relative luminescence units. Bars represent SD of quadruplicate wells. Data are representative of two independent experiments. (D) PHH lot BGW was cultured in 384-well plates before addition of 25 μM rifampicin in media on days 4 and 6 to induce CYP3A4 expression. At day 7 post-seed, CYP3A4 activity was measured by adding luciferin-IPA and a dilution series of 1-ABT in media. Fold change was calculated based on matching uninduced controls. Data are from one independent experiment.
Figure 5—figure supplement 2
ReFRAME hits re-confirmed in a P. vivax 12-day 1-aminobenzotriazole (1-ABT) assay.
(A) Hypnozonticidal potency comparison of 12 ReFRAME hits in 8- and 12-day 1-ABT dose–response confirmation assays. Cadralazine, plasmocid, and pidralazine potencies were unaffected by assay version, while MS-0735 was less potent, and poziotinib was more potent, in the 12-day 1-ABT assay. Budralazine, dramedilol, RGH-5526, dihydralazine, todralazine, endralazine, and mopidralazine were inactive (pEC50 <5) regardless of assay version. (B) Dose–response chart of poziotinib activity in the 12-day 1-ABT assay, pEC50 against hypnozoites = 6.05. Bars represent SEM.
Figure 5—figure supplement 3
Epigenetic inhibitor library screen and hits.
(A) Index chart of an epigenetic inhibitor library screened against P. vivax hypnozoites in a v3 (12-day 1-aminobenzotriazole [1-ABT]) assay. Teal circle: library, black square: DMSO, pink triangle: 200 nM nigericin. (B) Structures of epigenetic inhibitor hits which were confirmed to be active against P. vivax hypnozoites in dose–response assays; blue: histone deacetylase inhibitors.
Appendix 1—figure 1
Cytosine modification in P. vivax blood stages.
(A) P. vivax blood stages from patient isolates appeared negative when stained with 5mC. A white blood cell positive for 5mC serves as a stain control. (B) P. vivax blood stages from patient isolates appeared negative when stained with 5hmC. A white blood cell positive for 5hmC serves as a stain control. Bars represent 10 µm.
Tables
Table 1
Dose–response confirmation and counterscreens of primary screen hits and analogs.
Primary screen hits and structurally or mechanistically related compounds were tested by dose–response in 8 day P. vivax liver stage assays at Institute Pasteur of Cambodia and counterscreened against P. berghei liver schizonts, P. falciparum asexual blood stages of strain Dd2 and W2, and human cell lines HEK293T and HepG2. Values represent pEC50 or pCC50 ± SD of all independent experiments (n = 2–6) for which a pEC50 or pCC50 was obtained. An asterisk (*) indicates only one independent experiment resulted in a calculated pEC50 or pCC50. pEC50 is the inverse log of potency in M concentration, e.g. pEC50 3 = 1 mM, pEC50 6 = 1 μM, and pEC50 9 = 1 nM.
| Compound | Status | P. vivax hypnozoitesIPC | P. vivax liver schizontsIPC | Primary human hepatocytesIPC | P. berghei liver schizonts | P. falciparum asexual blood stage, strain Dd2 | Cytotoxicity, HEK293T | Cytotoxicity, HepG2 |
|---|---|---|---|---|---|---|---|---|
| (pEC50 ± SD) | (pEC50 ± SD) | (pCC50 ± SD) | (pEC50 ± SD) | (pEC50 ± SD) | (pCC50 ± SD) | (pCC50 ± SD) | ||
| Antihypertensives | ||||||||
| Cadralazine | Registered | 6.33 ± 0.29 | 6.33 ± 0.18 | < 5.00 | < 5.00 | < 4.90 | < 4.40 | 4.43* |
| Pildralazine | Discontinued | 6.08 ± 0.27 | ≤ 5.95 | < 5.00 | < 5.00 | < 4.90 | < 4.40 | 4.74* |
| Hydralazine | Registered | 5.75* | 5.42* | < 5.00 | < 5.00 | < 4.90 | < 4.40 | 4.51* |
| Budralazine | Registered | < 5.00 | < 5.00 | < 5.00 | 5.88 ± 0.4 | < 4.90 | < 4.40 | < 4.40 |
| Dihydralazine | Preclinical | < 5.00 | < 5.00 | < 5.00 | 5.53 ± 0.14 | 5.07 ± 0.07 | 4.7 ± 0.06 | 4.50 ± 0.11 |
| Endralazine | Discontinued | < 5.00 | < 5.00 | < 5.00 | < 5.00 | < 4.90 | 4.51* | 4.47* |
| Mopidralazine | Discontinued | < 5.00 | < 5.00 | < 5.00 | < 5.00 | < 4.90 | < 4.40 | < 4.40 |
| Todralazine | Unknown | < 5.00 | < 5.00 | < 5.00 | < 5.00 | < 4.90 | < 4.40 | < 4.40 |
| Dramedilol | Phase I | < 5.00 | < 5.00 | < 5.00 | < 5.00 | < 4.90 | 4.73 ± 0.06 | 4.60 ± 0.06 |
| RGH-5526 | Phase I | < | < 5.00 | < 5.00 | < 5.00 | < 4.90 | 4.87 ± 0.19 | 4.67 ± 0.12 |
| Anticancer | ||||||||
| Colforsin daropate | Registered | 7.07* | < 5.00 | < 5.00 | < 5.00 | < 4.90 | 4.71 ± 0.17 | 4.41* |
| Rhodamine 123 | Phase I | 5.23 ± 0.31 | ≤ 5.48 | < 5.00 | < 5.00 | 5.28 ± 0.08 | 5.28 ± 0.3 | 4.65 ± 0.07 |
| PAN-811 | Phase II | < 5.00 | < 5.00 | < 5.00 | 5.91 ± 0.29 | 5.66 ± 0.54 | 6.03 ± 0.23 | 5.77 ± 0.13 |
| Poziotinib | Phase II | < 5.00 | < 5.00 | < 5.00 | 5.23 ± 0.1 | 5.25 ± 0.03 | 5.27 ± 0.22 | 4.72 ± 0.16 |
| Other | ||||||||
| Narasin | Animal use | 5.79 ± 0.2 | 6.50* | < 5.00 | 9.09 ± 0.42 | 7.92 ± 0.13 | 7.57 ± 1.07 | 6.66 ± 0.58 |
| MS-0735 | Preclinical | 5.42* | ≤ 5.48 | < 5.00 | 6.22 ± 0.07 | 5.38 ± 0.09 | 6.07±0.22 | 6.05 ± 0.21 |
| Plasmocid | Discontinued | ≤ 5.48 | ≤ 5.95 | < 5.00 | 5.70 ± 0.27 | 6.74 ± 0.56 | 4.96 ± 0.14 | 4.95 ± 0.37 |
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Table 1—source data 1
Source data for Table 1.
- https://cdn.elifesciences.org/articles/98221/elife-98221-table1-data1-v1.xlsx
Table 2
Additional epigenetic inhibitors with activity against P. vivax liver stages.
| Epigenetic inhibitor | Target(s) | Hypnozoite pEC50 ± SD | Liver schizont pEC50 ± SD | PHH nuclei pCC50 ± SD |
|---|---|---|---|---|
| Panobinostat | HDAC | 6.98 ± 0.18 | 7.00 ± 0.15 | 5.68 ± 0.18 |
| AR42 | HDAC | 6.11 ± 0.24 | 6.30 ± 0.20 | 5.29 ± 0.27 |
| Raddeanin A | HDAC | 5.95 ± 0.00 | 5.38 ± 0.13 | 5.49 ± 0.02 |
| 666–15 | CREB | 5.88 ± 0.12 | 5.79 ± 0.03 | 5.46 ± 0.03 |
| Abexinostat | HDAC | 5.48 ± 0.00 | 5.26 ± 0.33 | < 5.00 |
| MI2 | Menin-MLL | 5.48 ± 0.00 | 5.48 ± 0.00 | < 5.00 |
| Givinostat | HDAC | 5.35 ± 0.45 | 5.35 ± 0.18 | < 5.00 |
| MMV019721 | P. falciparum ACS | 5.31 ± 0.03 | 5.25 ± 0.45 | < 5.00 |
| Cerdulatinib | SYK/JAK | 5.33 ± 0.20 | 5.26 ± 0.31 | < 5.00 |
| Pracinostat | HDAC | 5.32 ± 0.13 | 5.72 ± 0.20 | < 5.00 |
| CCT241736 | FLT3/Aurora Kinase | 5.24 ± 0.33 | 5.24 ± 0.34 | < 5.00 |
| Cyproheptadine | SETD | 5.24 ± 0.34 | 5.46 ± 0.03 | < 5.00 |
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HDAC: histone deacetylase. CREB: cAMP response element-binding protein. FLT3: fms-like tyrosine kinase 3. P. falciparum ACS: P. falciparum acetyl CoA synthetase. SYK: spleen tyrosine kinase. JAK: Janus kinase. SETD: SET domain containing histone lysine methyltransferase. Mean and standard deviation are from two or more independent experiments.
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Table 2—source data 1
Source data for Table 2.
- https://cdn.elifesciences.org/articles/98221/elife-98221-table2-data1-v1.xlsx
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens, female) | HEK293T | ATCC | ATCC cat:CRL-3216; RRID:CVCL_0063 | Transformed fetal cells |
| Cell line (Homo sapiens, male) | HepG2 | ATCC | ATCC cat:HB-8065; RRID:CVCL_0027 | Hepatoblastoma |
| Biological sample (Homo sapiens, male) | Primary human hepatocytes | BioIVT | Lot:UBV | Cryopreserved cryoplateable |
| Biological sample (Homo sapiens, male) | Primary human hepatocytes | BioIVT | Lot:BGW | Cryopreserved cryoplateable |
| Biological sample (Homo sapiens, male) | Primary human hepatocytes, female | BioIVT | Lot:QWK | Cryopreserved cryoplateable |
| Biological sample (Macaca fascicularis, male) | Primary simian hepatocytes | BioIVT | Lot:CWP | Cryopreserved cryoplateable |
| Biological sample (Macaca fascicularis, male) | Primary simian hepatocytes | BioIVT | Lot:NPI | Cryopreserved cryoplateable |
| Biological sample (Macaca fascicularis, male) | Primary simian hepatocytes | BioIVT | Lot:NDO | Cryopreserved cryoplateable |
| Biological sample (Macaca mulatta, male) | Primary simian hepatocytes | BioIVT | Lot:XXJ | Cryopreserved cryoplateable |
| Biological sample (Macaca mulatta, male) | Primary simian hepatocytes | BioIVT | Lot:NNF | Cryopreserved cryoplateable |
| Biological sample (An. dirus) | Mosquitoes | Shoklo Malaria Research Unit | Colony maintained on site | |
| Biological sample (An. dirus) | Mosquitoes | Institute Pasteur of Cambodia | Colony maintained on site | |
| Biological sample (An. dirus) | Mosquitoes | Armed Forces Research Institute of Medical Sciences | Colony maintained on site | |
| Biological sample (An. dirus) | Mosquitoes | University of Georgia | Colony maintained on site | |
| Biological sample (Anopheles stephensi) | Mosquitoes | University of Georgia | Colony maintained and infected at UGA, shipped to Calibr for P. berghei assays | |
| Biological sample (Plasmodium vivax) | Patient isolate | Shoklo Malaria Research Unit | PID:402389 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Shoklo Malaria Research Unit | PID:423955 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Shoklo Malaria Research Unit | PID:425583 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Shoklo Malaria Research Unit | PID:432054 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Shoklo Malaria Research Unit | PID:2020-013 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Shoklo Malaria Research Unit | PID:2020-014 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Institute Pasteur of Cambodia | PID:Pv593 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Institute Pasteur of Cambodia | PID:Pv595 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Institute Pasteur of Cambodia | PID:Pv602 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Institute Pasteur of Cambodia | PID:Pv603 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Institute Pasteur of Cambodia | PID:Pv606 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Institute Pasteur of Cambodia | PID:Pv608 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Institute Pasteur of Cambodia | PID:Pv609 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Institute Pasteur of Cambodia | PID:Pv611 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Institute Pasteur of Cambodia | PID:Pv623 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Institute Pasteur of Cambodia | PID:Pv624 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Institute Pasteur of Cambodia | PID:Pv635 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Institute Pasteur of Cambodia | PID:Pv640 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Institute Pasteur of Cambodia | PID:Pv644 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Institute Pasteur of Cambodia | PID:Pv708 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Institute Pasteur of Cambodia | PID:Pv836 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Institute Pasteur of Cambodia | PID:Pv838 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Institute Pasteur of Cambodia | PID:Pv846 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Institute Pasteur of Cambodia | PID:Pv847 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Institute Pasteur of Cambodia | PID:Pv849 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Institute Pasteur of Cambodia | PID:Pv893 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Institute Pasteur of Cambodia | PID:Pv922 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Institute Pasteur of Cambodia | PID:Pv923 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Institute Pasteur of Cambodia | PID:Pv950 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Institute Pasteur of Cambodia | PID:Pv951 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Institute Pasteur of Cambodia | PID:Pv952 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Institute Pasteur of Cambodia | PID:Pv959 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Institute Pasteur of Cambodia | PID:Pv1014 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Institute Pasteur of Cambodia | PID:Pv1020 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Institute Pasteur of Cambodia | PID:Pv1024 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Institute Pasteur of Cambodia | PID:IV21-075 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Institute Pasteur of Cambodia | PID:PQRC21-113 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Institute Pasteur of Cambodia | PID:PQRC21-135 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium vivax) | Patient isolate | Mahidol Vivax Research Unit | PID:VTTY201 | Fresh isolate fed to An. dirus mosquitoes |
| Biological sample (Plasmodium cynomolgi) | M/B strain | PMID:31536608 | Emory National Primate Research Center | |
| Biological sample (Plasmodium cynomolgi) | Rossan strain | PMID:18788885 | Emory National Primate Research Center | |
| Biological sample (Plasmodium cynomolgi) | B strain | PMID:32660993 | Armed Forces Research Institute of Medical Sciences | |
| Biological sample (Plasmodium cynomolgi) | P. berghei ANKA strain GFP Lucama1-eef1a (line 1052cl1) | PMID:36100902 | University of Georgia | |
| Biological sample (Plasmodium falciparum) | Dd2-HLH | BEI Resources | Cat#:MRA-156 | |
| Biological sample (Plasmodium cynomolgi) | DC | This paper | University of Georgia | |
| Biological sample (Plasmodium falciparum) | W2 | PMID:7729473 | University of Georgia | |
| Strain, strain background (Macaca fuscata, male) | Monkey, used for experimental animal infection | Emory National Primate Research Center | Not genetically modified | |
| Strain, strain background (Macaca fuscata, male) | Monkey, used for experimental animal infection | Emory National Primate Research Center | Not genetically modified | |
| Antibody | anti P. vivax Upregulated in Infectious Sporozoites 4 (rPvUIS4) (recombinant mouse monoclonal) | PMID:30333026 | IFA (1:10,000) | |
| Antibody | anti-PvMIF (rabbit polyclonal) | PMID:25800544 | IFA (1:1000) | |
| Antibody | anti-PcHSP70 (rabbit polyclonal) | This paper | n/a | IFA (200 ng/ml) |
| Antibody | anti-PcUIS4 (mouse monoclonal) | This paper | n/a | IFA (10 ng/ml) |
| Antibody | anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Goat monoclonal) | Thermo Fisher Scientific | Cat#: A-11001; RRID:AB_2534069 | IFA (1:1000) |
| Antibody | anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Goat monoclonal) | Thermo Fisher Scientific | Cat#:A11013; RRID:AB_2534080 | IFA (1:1000) |
| Antibody | anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 (Goat monoclonal) | Thermo Fisher Scientific | Cat#:A11036; RRID:AB_10563566 | IFA (1:1000) |
| Antibody | 5-Methylcytosine Recombinant Antibody (rabbit monoclonal) | Thermo Fisher Scientific | Cat#:MA5-24694: RRID:AB_2665309; Clone:RM231 | 10 μg/ml |
| Antibody | 5-Hydroxymethylcytosine Recombinant Antibody (rabbit monoclonal) | Thermo Fisher Scientific | Cat#:MA5-24695; RRID:AB_2665308; Clone:RM236 | 10 μg/ml |
| Antibody | anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Texas Red (goat monoclonal) | Thermo Fisher Scientific | Cat#:T-2767; RRID:AB_2556776 | 10 μg/ml |
| Antibody | anti-Plasmodium GAPDH (mouse monoclonal) | European Malaria Reagent Repository | Cat#:13.3 | 100 ng/ml |
| Software, algorithm | Genedata Screener, Version 15.0.1-Standard | Genedata | ||
| Chemical compound, drug | Budralazine | Chemcruz | Cat3:sc-504334 | Batch D3019 |
| Chemical compound, drug | Cadralazine | Chemcruz | Cat#:sc-500641 | Batch B24217 |
| Chemical compound, drug | Hydralazine | Selleckchem | Cat#:S2562 | Batch S256202 |
| Chemical compound, drug | Dihydralazine | Calibr at Scripps | Code:CBR-001-571-820-4 | |
| Chemical compound, drug | Plasmocid | Calibr at Scripps | Code:CBR-001-572-110-5 | |
| Chemical compound, drug | MS-0735 | Calibr at Scripps | Code:CBR-001-572-134-3 | |
| Chemical compound, drug | Hydralazine | Calibr at Scripps | Code:CBR-001-572-134-3 | |
| Chemical compound, drug | Colforsin daropate | Calibr at Scripps | Code:CBR-001-586-408-1 | |
| Chemical compound, drug | PAN-811 | Calibr at Scripps | Code:CBR-001-586-749-9 | |
| Chemical compound, drug | Todralazine | Calibr at Scripps | Code:CBR-001-586-916-6 | |
| Chemical compound, drug | RGH-5526 | Calibr at Scripps | Code:CBR-001-587-032-3 | |
| Chemical compound, drug | Budralazine | Calibr at Scripps | Code:CBR-001-587-246-5 | |
| Chemical compound, drug | Dramedilol | Calibr at Scripps | Code:CBR-001-593-286-2 | |
| Chemical compound, drug | Endralazine | Calibr at Scripps | Code:CBR-001-597-262-0 | |
| Chemical compound, drug | Cadralazine | Calibr at Scripps | Code:CBR-001-624-776-0 | |
| Chemical compound, drug | Pildralazine | Calibr at Scripps | Code:CBR-001-635-378-9 | |
| Chemical compound, drug | Mopidralazine | Calibr at Scripps | Code:CBR-001-635-852-4 | |
| Chemical compound, drug | Rhodamine 123 | Calibr at Scripps | Code:CBR-050-127-020-8 | |
| Chemical compound, drug | Narasin | Calibr at Scripps | Code:CBR-050-127-705-0 | |
| Chemical compound, drug | Poziotinib | Calibr at Scripps | Code:CBR-001-574-260-6 | |
| Chemical compound, drug | Panobinostat | Targetmol | Cat#:T2383 | |
| Chemical compound, drug | Abexinostat | Targetmol | Cat#:T0431 | |
| Chemical compound, drug | Pracinostat | Targetmol | Cat#:T1890 | |
| Chemical compound, drug | Cyproheptadine | Targetmol | Cat#:T0174 | |
| Chemical compound, drug | Cerdulatinib | Targetmol | Cat#:T2487 | |
| Chemical compound, drug | MI2 | Targetmol | Cat#:T2649 | |
| Chemical compound, drug | Raddeanin A | Targetmol | Cat#:T3878 | |
| Chemical compound, drug | CCT241736 | Targetmol | Cat#:T4428 | |
| Chemical compound, drug | 666-15 | Targetmol | Cat#:T5318 | |
| Chemical compound, drug | Givinostat | Targetmol | Cat#:T6279 | |
| Chemical compound, drug | AR42 | Targetmol | Cat#:T6392 | |
| Chemical compound, drug | MMV019721 | Medicines for Malaria Venture | Code:MMV019721 | Batch:MMV019721-08, MMV019721-10 |
| Chemical compound, drug | MMV084978 | Medicines for Malaria Venture | Code:MMV084978 | Batch:MMV084978-04, MMV084978-05 |
| Chemical compound, drug | 5-Azacytidine | Cyamen Chem | Cat#:11164 | |
| Chemical compound, drug | 1-Aminobenzotriazole | Cyamen Chem | Cat#:15252 | |
| Commercial assay or kit | Cell-Titer Glo | Promega | Cat#:G7573 | |
| Commercial assay or kit | EpiQuik DNA Methyltransferase (DNMT) Activity/Inhibition Assay Kit | EpiGentek | Cat#:P-3010 | |
| Commercial assay or kit | Epitect fast bisulfite conversion kit | QIAGEN | Cat#:59824 | |
| Commercial assay or kit | CYP3A4 luciferin-IPA kit | Promega | Cat#:V9001 | Used Lytic protocol |
Additional files
-
MDAR checklist
- https://cdn.elifesciences.org/articles/98221/elife-98221-mdarchecklist1-v1.docx
-
Supplementary file 1
Summary of ReFRAME plate (40 plates labeled 1–41, with 37 skipped) run metrics including average hypnozoites and schizont counts per well, Z′ factor for 1 μM monensin wells, screening location (Shoklo Malaria Research Unit, Thailand, or Pasteur Institute of Cambodia) primary human hepatocyte (PHH) lot used, and P. vivax patient isolate used.
Due to an error during library plating, some plates contained only 1 well of monensin, preventing calculation of a Z′ factor for those plates (listed as N.A.).
- https://cdn.elifesciences.org/articles/98221/elife-98221-supp1-v1.docx
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Supplementary file 2
Potency data (pEC50) for select ReFRAME hits against P. cynomolgi liver forms assayed at NITD in primary simian hepatocyte (PSH) lots NDO, NPI, XXJ infected with one batch of P. cynomolgi sporozoites.
Cytotoxicity (pCC50) against PSH was measured using nuclei counts. Maduramicin is a positive control with activity against P. cynomolgi hypnozoites.
- https://cdn.elifesciences.org/articles/98221/elife-98221-supp2-v1.docx
-
Supplementary file 3
Summary statistics of read sets, percentage of mapped reads, read methylation levels, conversion rate, and genome-wide methylation levels from bisulfite sequencing.
- https://cdn.elifesciences.org/articles/98221/elife-98221-supp3-v1.docx
-
Supplementary file 4
Pharmacokinetic data report from Wuxi for cadralazine in Rhesus macaques.
- https://cdn.elifesciences.org/articles/98221/elife-98221-supp4-v1.xlsx
-
Supplementary file 5
Contents of the Targetmol Epigenetic Library.
- https://cdn.elifesciences.org/articles/98221/elife-98221-supp5-v1.xlsx
-
Supplementary file 6
Usage of reagents for experiments and replication.
- https://cdn.elifesciences.org/articles/98221/elife-98221-supp6-v1.xlsx
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A drug repurposing approach reveals targetable epigenetic pathways in Plasmodium vivax hypnozoites
eLife 13:RP98221.
https://doi.org/10.7554/eLife.98221.2
