Figures and data
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 factor). Teal: library, black: DMSO, red: 1 μM monensin. (B) Primary screen hits were confirmed by dose-response in 8-day P. vivax liver stage assays and counterscreened against P. berghei liver schizonts, P. falciparum asexual blood stages (strain Dd2), HEK293T, and HepG2. Values represent pEC50 or pCC50 ± SD of all independent experiments (n=2-6) for which a pEC50 or pCC50 was obtained. (C) Dose-response curves for cadralazine against P. vivax and P. cynomolgi liver forms in 8-day assays at the IPC, UGA, and NITD. (B,C) Heat maps represent red as more potent and blue as inactive at highest dose tested. 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). (C) 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.
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.
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 <.0001, for schizonts H(7) = 88.66, p <.0001, with Dunn’s multiple comparisons, *p <.05, ***p <.001, ****p <.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.
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 retrieved22 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.
Additional epigenetic inhibitors with activity against P. vivax liver stages.
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.
ReFRAME screen run details.
(A) Table of each ReFRAME plate (40 plates labelled 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) 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.). (B) Simple linear regression correlating Z’ factor with average hypnozoite count per well. (C) Index chart from Fig. 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.
Dose-response confirmation and counterscreens of primary screen hits and analogs.
(A) 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, P. cynomolgi asexual blood stages, 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. (B) 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.
Select ReFRAME hits confirmed at Novartis Institute for Tropical Diseases (NITD).
(A) 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. (B) Potency data (pEC50) for 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 (A,B) Heat maps represent red as more potent and blue as inactive at highest dose tested.
Pharmacokinetics of cadralazine in nonhuman primates.
Mean plasma concentration of cadralazine was measured in three males 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.
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.
Cytosine modifications in P. vivax liver forms, full panels from Case 1 (also shown in Fig. 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.
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.
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.
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, 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 Case 1, 2, and 3 in Fig. 3. Hypnozoites were classified as forms with an area 125µm2 and smaller.
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 20µm. (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 determined using Kruskal-Wallis tests for hypnozoites and schizonts, with Dunn’s multiple comparisons, ****p <.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 and B are from Experiment 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. (C) Summary statistics of read sets, percentage of mapped reads, read methylation levels, conversion rate and genome-wide methylation levels from bisulfite sequencing.
Cytosine and methylation density plots for P. vivax sporozoites.
(A) CG content of chromosome 1 to 14 (Chr 1-14). The total number of cytosines quantified on each strand using 1 kbp long non-overlapping windows. (B) The total number of methylated cytosines quantified on each strand using 1 kbp long non-overlapping windows.
Cytosine and methylation density plots for P. cynomolgi sporozoites.
(A) CG content of chromosome 1 to 14 (Chr 1-14). The total number of cytosines quantified on each strand using 1 kbp long non-overlapping windows. (B) The total number of methylated cytosines quantified on each strand using 1 kbp long non-overlapping windows.
Monensin activity in all control wells based on 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 <.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.
Characterization of primary human hepatocyte (PHH) metabolism following 1-aminobenzotriazole (1-ABT) treatment.
(A) PHH lot BGW were seeded into 384-well plates and cultured for 7 days before addition of a dilution series of 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. (B) PHH lot BGW were 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. (C) PHH lot BGW were seeded in 384-well plates and cultured for 7 days before treatment with 100 μM 1-ABT for 1h, followed by addition of substrates for 1 h 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 <.0001, ***p <.001, **p <.01, ns, not significant.
ReFRAME hits re-confirmed in a P. vivax 12-day 1-ABT assay.
(A) Hypnozonticidal potency comparison of 12 ReFRAME hits in 8-day 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.
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-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.
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.
