A Drug Repurposing Approach Reveals Targetable Epigenetic Pathways in Plasmodium vivax Hypnozoites

Radical cure of Plasmodium vivax malaria must include elimination of quiescent ‘hypnozoite’ forms in the liver; however, the only FDA-approved treatments are contraindicated in many vulnerable populations. To identify new drugs and drug targets for hypnozoites, we screened the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) library and a collection of epigenetic inhibitors against P. vivax liver stages. From both libraries, we identified inhibitors targeting epigenetics pathways as selectively active against P. vivax and P. cynomolgi hypnozoites. These include DNA methyltransferase (DNMT) inhibitors as well as several inhibitors targeting histone post-translational modifications. Immunofluorescence staining of Plasmodium liver forms showed strong nuclear 5-methylcystosine signal, indicating liver stage parasite DNA is methylated. Using bisulfite sequencing, we mapped genomic DNA methylation in sporozoites, revealing DNA methylation signals in most coding genes. We also demonstrated that methylation level in proximal promoter regions as well as in the first exon of the genes may affect, at least partially, gene expression in P. vivax. The importance of selective inhibitors targeting epigenetic features on hypnozoites was validated using MMV019721, an acetyl-CoA synthetase inhibitor that affects histone acetylation and was previously reported as active against P. falciparum blood stages. In summary, our data indicate that several epigenetic mechanisms are likely modulating hypnozoite formation or persistence and provide an avenue for the discovery and development of improved radical cure antimalarials.

ReFRAME library was screened using the original, 8-day radical cure assay, in which developing liver schizonts and mature, PI4Ki-insensitive hypnozoites were treated on days 5-7 post-infection (7,13). On treatment days, a pintool was used to transfer 40 nL of compounds from the source plates to the assay plates. A single PHH lot, UBV, was first used for screening, however, once all available cryovials were used, screening was completed with lot BGW (fig. S1). Screening was 5 initiated at SMRU until a second screening site was established at IPC, where all unfinished source plates were shipped and assayed. Some plates were assayed more than once in order to obtain a single run with a sufficient Z' factor of > 0.0 or two moderate-quality runs allowing for identification of reproducibly-active wells ( fig. S1). Quantification of PvLS growth was performed by fixing and staining cultures with recombinant mouse-anti P. vivax Upregulated in 10 Infectious Sporozoites 4 (rPvUIS4) (42). followed by high-content imaging and analysis using an ImageXpress Micro (Molecular Devices) or Lionheart FX (Agilent). Hypnozoites were classified as forms of less than 125 μm 2 growth area.
Normalization, hit selection, and dose-response confirmation in P. vivax liver stage assays 15 Primary screening data were imported into Genedata Screener, Version 15.0.1-Standard and normalized to DMSO (neutral) and inhibitor (monensin) control-treated wells (neutral controls minus inhibitors). For four plates where the monensin control failed due to solubility issues combined with PHH lot variability (fig. S12), data were normalized using the Robust Z-Score 20 method, which calculates for each well the Robust Z-Score (number of standard deviations off the median) based on the statistics of the compound wells per plate. Genedata multiplicative pattern correction was applied to adjust for plate edge effects. Sixty-two most active (≥ 67% normalized inhibition of hypnozoite numbers) and non-toxic (≤ 40% host cell toxicity) compounds and 10 hydrazinophthalazines were selected for reconfirmation in an 8-point 1:3 dose response following 25 the 8-day protocol with PHH lot BGW using a dose-response of monensin and nigericin as redundant positive controls. Once hydralazine and cadralazine were identified as reconfirmed hits, commercially available batches of powder were obtained (budralazine, Chemcruz cat sc-504334 batch D3019, cadralazine, Chemcruz cat sc-500641 batch B2417, and hydralazine, Selleckchem cat s2562 batch S256202) and used for additional reconfirmation runs using the same 8-day 30 protocol ( Fig. 1 and fig. S2).
Hit confirmation in P. cynomolgi liver stage assays at UGA P. cynomolgi assays at UGA were performed using the step-by-step protocol for the P. vivax liver 35 stage assay (41) with a few modifications. A Japanese macaque (Macaca fuscata) was intravenously infected with P. cynomolgi Rossan strain cryopreserved ring stage parasites (43) and allowed to reach patency. When parasitemia reached approximately 5,000 parasites per microliter, An. dirus mosquitoes were fed directly on the infected animal over a period of three to four days. The blood-fed mosquitoes were then checked for infection six to eight days by dissecting and 40 staining midguts with 2% mercurochrome to detect oocysts. Two experiments were performed, one with PSH lot CWP, and one with PSH lot NPI. Two days after assay plates (Greiner Bio-One cat 781956) were seeded with 20,000 live PSH per well, sporozoites were dissected from mosquito salivary glands at day 16 post-bloodmeal and allowed to infect cultures. Hits were confirmed using the same 8-day radical cure assay. On treatment days, a pin tool was used to transfer 40 nL of (http://www.malariaresearch.eu) followed by high-content imaging and analysis using an ImageXpress Micro (Molecular Devices). Hypnozoites were classified as forms of less than 105 μm 2 growth area.
Hit confirmation in P. cynomolgi and P. vivax liver stage assays at NITD 5 Lots of both PSH and PHH were obtained from BioIVT. Hepatocytes were seeded at 22,000 cells per well in a 384-well plate (Corning cat 356667). Prior to and during the infection, the hepatocytes were cultured in BioIVT CP Medium (cat Z990003) with the addition of 1% PSN (Gibco cat 15640055) and 0.1 % gentamicin in the case of P. vivax. Two days post-seeding, the hepatocytes 10 were infected with sporozoites dissected from the salivary glands of An. dirus mosquitoes. Sporozoites were collected in RPMI 1640 (KD Medical cat CUS-0645). Hepatocytes were infected with 10,000 sporozoites per well and spun for 5 minutes at 200 × g. Once the sporozoites were removed after 24 h of incubation, the culture media was exchanged to include 5% PSN in the case of P. cynomolgi. On days 4, 5, 6, and 7 post-infection, the hepatocytes received fresh compound 15 addition in media. The cells were fixed on day 8 using 4% paraformaldehyde.
Liver stage parasites were detected by immunofluorescence assay. Hepatocytes were permeabilized for 1 h at room temperature in blocking buffer consisting of 2% Bovine Serum Albumin (Millipore Sigma cat A2153) and 0.2% Triton X-100 (Millipore Sigma cat 648466) in 20 1× PBS (Gibco cat 20012-027). For P. cynomolgi staining, the two in-house primary antibodies used were mouse anti-PcUIS4 monoclonal at 10 ng/mL, and rabbit anti-PcHSP70 polyclonal at 200 ng/mL. For P. vivax staining, rabbit anti-PvMIF was used at 1:1000 (44). The primary antibodies were diluted in blocking buffer and incubated overnight at 4 °C. Hepatocytes were washed thrice with 1× PBS and then incubated with secondary antibodies (Invitrogen cat A11013, 25 RRID: AB_2534080 and A11036, RRID: AB_10563566) used at a 1:1000 dilution and Hoechst 33342 (Invitrogen cat H3570) used at 2 μg/mL for 2 h at room temperature. After the incubation, the hepatocytes were washed 3 times with 1× PBS and were stored in 50 μL per well of 1× PBS prior to imaging on an ImageXpress Micro (Molecular Devices).

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Confirmed hit counterscreens: P. falciparum asexual blood stage at Calibr The SYBR Green I-based parasite proliferation assay (45) was used to determine the activity of compounds against the asexual blood stage of P. falciparum strain Dd2-HLH, a transgenic line expressing firefly luciferase (46). Briefly, acoustic compound transfer (Labcyte Echo 550) was 35 used to prepare assay ready plates to which parasites in assay medium were added and incubated with compounds for 72 h. SYBR Green I in lysis buffer was used as detection reagent. Fluorescence signal was read on the PHERAstar FSX plate reader (BMG Labtech). Compounds were tested in technical triplicates on different assay plates across three biological replicates performed on different days. Data were uploaded to Genedata Screener, Version 16.0.3-Standard 40 and normalized to DMSO (neutral) and inhibitor control-treated wells (neutral controls minus inhibitors), with 1.25 µM dihydroartemisinin used as a positive control. Dose curves (thirteen point, 1:3 dilution series) were fitted with the four parameter Hill Equation.
Confirmed hit counterscreens: P. falciparum asexual blood stage at UGA 45 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 31, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023 Budralazine, cadralazine and hydralazine (same catalog and batches as above) were tested using the [ 3 H]-hypoxanthine drug susceptibility assay as previously described, with some modifications (47). Strain W2 (48, 49) was grown in continuous culture using RPMI 1640 media containing 10% heat-inactivated type A+ human plasma, sodium bicarbonate (2.4 g/L), HEPES (5.94 g/L), and 4% washed human type A+ erythrocytes. Cultures were gassed with a 90% N2, 5% O2, and 5% CO2 5 mixture and incubated at 37 °C. Cultures were sorbitol synchronized to achieve >70% ring stage parasites (50). Assay were started by establishing a 0.5−0.7% parasitemia and 1.5% hematocrit in complete media. Assays were performed in 96-well plates with a volume of 90 μL/well of parasitized erythrocytes and 10 μL/well of 10× test compound. Dihydroartemisinin was plated as a positive control and DMSO as a negative control. Assay plates were incubated in the above-10 mentioned gas mixture at 37 °C for 48 h; then, 3 H-hypoxanthine (185 MBq, Perkin Elmer cat NET177005MC) was added, and plates were incubated for another 24 h. After 72 h of incubation, the assay plates were frozen at −80 °C. Plates were allowed to thaw at room temperature before well contents were collected onto filtermats using a plate harvester (PerkinElmer). A Micro Beta liquid scintillation counter (PerkinElmer) was used to quantify radiation (counts-per-minute) 15 representing relative parasite growth. Values were normalized to controls and plotted using CDD Vault. Potency values represent means of at least two independent experiments.
Confirmed hit counterscreens: P. cynomolgi asexual blood stage at UGA 20 Budralazine, cadralazine and hydralazine (same catalog and batches as above) were tested against P. cynomolgi DC strain using the [ 3 H]-hypoxanthine drug susceptibility assay as previously described, with some modifications (47). P. cynomolgi was grown in continuous culture using RPMI 1640 +GlutaMAX media containing 20% heat-inactivated rhesus serum, hypoxanthine (32 mg/L), HEPES (7.15 g/L), additional glucose (2g/L), and 5% washed rhesus erythrocytes. Cultures 25 were incubated at 37°C under mixed gas conditions of 90% N2, 5% O2, and 5% CO2. Schizonts were synchronized over a 60/20 Percoll gradient to achieve >90% late-stage parasites. Assays were started the following day when ring-stage parasites were present. Parasites were prepped for assay by establishing 0.5% ring-stage parasitemia and 2% hematocrit in complete media without hypoxanthine. Assays were performed in 96-well plates with a volume of 90 μL/well of parasitized 30 erythrocytes and 10 μL/well of 10× test compounds. Compounds were plated from a starting concentration of 5 μM in an 11-point 1:2 dilution series and tested in duplicate. Uninfected RBCs were plated as a positive control and DMSO was used as a negative control. [ 3 H]-hypoxanthine (185 MBq, Perkin Elmer cat NET177005MC) was then added to all wells and plates were incubated under the previously-mentioned conditions for 72 h. After 72 h the assay plates were 35 frozen at -80 °C. Plates were thawed the following day at room temperature and well contents were collected onto filtermats using a plate harvester (PerkinElmer). A Micro Beta liquid scintillation counter (PerkinElmer) was used to quantify radiation (counts-per-minute) representing relative parasite growth. Values were normalized to controls and plotted using CDD Vault. Potency values represent means of at least two independent experiments.

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Confirmed hit counterscreens: P. berghei liver stage at Calibr For P. berghei liver stage assays, a colony of An. stephensi mosquitoes was maintained in the UGA Sporocore using methods previously described (51). In summary, adults were fed 5% dextrose 45 (w/v) and 0.05% para-aminobenzoic acid (w/v) soaked into cotton pads and kept at a temperature of 27 °C, relative humidity of 75-85%, and a 12 h light/dark cycle. PbGFP-LUCCON sporozoites . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 31, 2023. ; https://doi.org/10.1101/2023.01.31.526483 doi: bioRxiv preprint were produced as previously described (51). In summary, female C57BL/6 or Hsd:ICR(CD-1) mice (Envigo) were injected intraperitoneally with 5×10 6 -5×10 7 blood stage parasites in 500 μL PBS 3-4 days before mosquito infections. Once parasitemia reached 2-6%, mice were anesthetized with 0.5 mL 1.25% 2,2,2-Tribromoethanol (v/v, Avertin, Sigma-Aldrich) and placed on top of cage of An. stephensi mosquitoes (3-7 days post-emergence) for 20 min to serve as an infectious 5 bloodmeal. Infected mosquitoes were shipped to Calibr, where sporozoites were dissected out of mosquito salivary glands and used for luciferase-based infection assay as previously described (52). Briefly, HepG2 cells (ATCC cat HB-8065, RRID: CVCL_0027) were infected with freshly dissected sporozoites. The infected cells were incubated with compounds of interest in 1536-well plates for 48 hours, and intracellular parasite growth was measured using bioluminescence.

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Compounds were tested in technical triplicates on different assay plates across three biological replicates performed on different days. Data were uploaded to Genedata Screener, Version 16.0.3-Standard and normalized to DMSO (neutral) and inhibitor control-treated wells (neutral controls minus inhibitors), with 1 µM KAF156 used as a positive control. Dose curves (thirteen point, 1:3 dilution series) were fitted with the four parameter Hill Equation.

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Immunofluorescent staining of methyl-cytosine modifications in P. vivax liver stages Sporozoites from three different P. vivax cases were infected into PHH lot BGW at day 2 postseed (for case 1) or day 3 post-seed (for cases 2 and 3) in 384-well plates (Greiner Bio-One cat 781956) using the same methods for initiating P. vivax liver stage screening assays described 35 above. Cultures were fixed at day 6 post-infection and stained with rPvUIS4 and Hoechst 33342 as previously described (41,42). Cultures were then stained for either with rabbit anti-5mC monoclonal antibody (clone RM231, ThermoFisher Scientific cat MA5-24694, RRID: AB_2665309) or rabbit anti-5hmC monoclonal antibody (clone RM236, ThermoFisher Scientific cat MA5-24695, RRID: AB_2665308) using methods adapted from those previously described 40 (18). In summary, cultures were re-permeablilized with 0.1% (v/v) Triton-X 100 for 20 min at room temperature and then washed thrice with 1× PBS. Chromatin was then denatured with 4 N HCl for 30 min at room temperature and washed thrice with 1× PBS. The denaturing reaction was then neutralized with 100 mM Tris (pH 8.0) for 10 min at room temperature and washed thrice with 1× PBS. Cultures were then quenched with 50 mM NH4Cl for 10 min at room temperature 45 and washed thrice with 1× PBS. Cultures were then blocked with 0.1% (v/v) Tween 20 and 2% (w/v) Bovine Serum Albumin for 10 min at room temperature and washed thrice with PBS.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 31, 2023. ; https://doi.org/10. 1101/2023 Cultures were then stained with either antibody diluted to 10 μg/mL in PBS overnight at 4 °C and washed thrice with 1× PBS. Cultures were then stained with 10 μg/mL Texas Red-conjugated, goat anti-rabbit IgG secondary antibody (ThermoFisher Scientific, cat T-2767, RRID: AB_2556776) overnight at 4 °C and washed thrice with 1× PBS. For a negative stain control, a separate set of infected wells were prepared as above and stained with secondary antibody only 5 (2' control, Fig. 2B). High resolution images of individual PvLS parasites and PHH nuclei were obtained by capturing 8 planes in the Z dimension using a 100× objective on Deltavision Core (GE Healthcare Life Sciences) and deconvoluted using softWoRx (GE Healthcare Life Sciences) ( Fig. 2A and figs. S4, S5, and S6). An ImageXpress Micro high content imager was used to quantify methyl-cytosine modifications for the entire population of PvLS parasites from each case.

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A 20× objective was used to capture 25 fields of view from each well (covering the entire growth area) of the 384-well plate. Using the associated MetaXpress high content analysis software, the rPvUIS4 stain from each PvLS parasite was used to define parasite objects, and the 5mC or 5hmC staining of host cell nuclei were used to define positive methyl-cytosine modification objects. The 2-dimensional area of intersection of both objects was then quantified for each parasite (Fig. 2B, Immunofluorescent staining of methyl-cytosine modifications in P. cynomolgi liver stages A Japanese macaque (Macaca fuscata) was intravenously infected with P. cynomolgi M/B strain 20 (53) and allowed to reach patency before skin feeding to An. dirus mosquitoes as described above. One round of mosquito dissection and culture infection was performed with PSH lot NPI. Two days after assay plates (Greiner Bio-One cat 781956) were seeded with 20,000 primary simian hepatocytes per well, sporozoites were dissected from mosquito salivary glands at day 16 postbloodmeal and allowed to infect cultures. Cultures were fixed on day 8 post-infection and stained 25 for 5mC and 5hmC as described above. An ImageXpress Micro high content imager was used to quantify methyl-cytosine modifications for the entire population of P. cynomolgi liver stage parasites. A 20× objective was used to capture 25 fields of view from each well (covering the entire growth area) of the 384-well plate. Using the associated MetaXpress high content analysis software, the GAPDH stain from each liver stage parasite was used to define parasite objects, and 30 the 5mC or 5hmC staining of host cell nuclei were used to define positive methyl-cytosine modification objects. The 2-dimensional area of intersection of both objects was then quantified for each parasite ( fig. S8).
Collection of P. vivax and P. cynomolgi sporozoites for methyl-cytosine characterization 35 For quantification of methyl-cytosine modification levels by mass spec, sporozoites from 3 different P. vivax cases, numbering 18.7×10 6 from case 1, 10×10 6 from case 2, and 14.7×10 6 from case 3, were dissected from infected An. dirus mosquitoes at IPC as previously described (41) and cryopreserved as previously described (54). For quantification of DMNT activity from nuclear 40 extracts, sporozoites from 2 different P. vivax cases, numbering 21×10 6 from case 1 and 20×10 6 from case 2, were similarly dissected and cryopreserved. To serve as a negative control, salivary glands from uninfected mosquitoes at IPC were similarly dissected and cryopreserved. A total of 4.8×10 6 sporozoites for mass spec and 34.1×10 6 sporozoites for DNMT activity assays were also collected from An. dirus mosquitoes infected from feeding on a rhesus macaque infected with P. 45 cynomolgi M/B strain at ENPRC and cryopreserved as described above. To serve as a negative control, salivary glands and ovaries from uninfected mosquitoes at ENPRC were similarly . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 31, 2023. ; https://doi.org/10.1101/2023.01.31.526483 doi: bioRxiv preprint dissected and cryopreserved. For mapping of methyl-cytosine modifications by bisulfite sequencing, sporozoites from three different P. vivax cases, numbering 9.8×10 6 from case 1, 12.3×10 6 from case 2, and 15.1×10 6 from case 3, were dissected from infected An. dirus mosquitoes at IPC and cryopreserved as described above. A total of 5.3×10 6 sporozoites were also collected from An. dirus mosquitoes infected from feeding on a rhesus macaque infected with P. 5 cynomolgi M/B strain at ENPRC and cryopreserved as described above. Frozen sporozoites and salivary glands were shipped from IPC and ENPRC to University of California, Riverside on dry ice.

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Parasite pellets were lysed with 100 µl lysis buffer (20 mM Tris, pH 8.1, 20 mM EDTA, 400 mM NaCl, 1% SDS and 20 mg/ml proteinase K) and incubated at 55 0 C overnight. Saturated solution of NaCl (0.5× volume of reaction mixture) was subsequently added to the digestion mixture and incubated at 55 °C for another 15 min. The samples were centrifuged at 14500 RCF for 30 min at 15 4 °C and the supernatant removed to a 1.5-mL microcentrifuge, genomic DNA (gDNA) was then precipitated with 2× volume of 100% chilled ethanol and resuspended in 95 μL water. Samples were then treated with 3 μL of 10 mg/mL RNase A and 2 μL of 25 units/μL RNase T1 and incubated overnight at 37 °C. gDNA was then extracted by chloroform/isoamyl alcohol solution, precipitated again with 100% chilled ethanol, and washed with 70% ethanol. The gDNA pellets 20 were then dissolved in nuclease-free water. One μg of gDNA was enzymatically digested into mononucleosides using nuclease P1 and alkaline phosphatase. Enzymes in the digestion mixture were removed by chloroform extraction. The resulting aqueous layer was dried by using a Speedvac and the dried residues were subsequently reconstituted in doubly distilled water. Approximately 5 ng of the DNA digestion mixture was injected for LC-MS/MS/MS analyses for 25 quantifications of 5mC, 5hmC and dG. An LTQ XL linear ion-trap mass spectrometer equipped with a nano electrospray ionization source and coupled with an EASY-nLC II system (Thermo Fisher Scientific) was used for the LC-MS/MS/MS experiments. The amounts of 5mC, 5hmC and dG (in moles) in the nucleoside mixtures were calculated from area ratios of peaks found in the selected-ion chromatograms for the analytes over their corresponding isotope-labeled standards, 30 the amounts of the labeled standards added (in moles), and the calibration curves. The final levels of 5mC and 5hmC, in terms of percentages of dG, were calculated by comparing the moles of 5mC and 5hmC relative to those of dG.

Extraction of nuclear protein extracts 35
Cryopreserved sporozoites, or parasites extracted from red blood cells by saponin lysis, were resuspended in 1 ml of cytoplasmic lysis buffer (20mM HEPES pH 7.9, 10 mM KCl, 1mM EDTA, 1mM EGTA, 1mM dithiothreitol (DTT), 0.5 mM AEBSF, 0.65% Igepal, 1× Roche complete protease inhibitor cocktail) and incubated for 10 min on ice. Nuclei were separated from 40 cytoplasmic fraction by 10 min of centrifugation at 1500 RCF followed by two washes with cytoplasmic lysis buffer and one time wash with ice cold 1x PBS. Nuclei pellets were resuspended in 100 µl of nuclei lysis buffer (20 mM HEPES pH 7.9, 0.1 M NaCl, 1mM EDTA, 1 mM EGTA, 1mM DTT, 25 % glycerol, 0.5 mM AEBSF, 1× Roche complete protease inhibitor cocktail) for 20 min at 4 °C with rotation. Nuclear extracts were cleared by 10 min of centrifugation at 6000 45 RCF. Protein concentration of nuclear extract was quantified by BCA assay and DNMT assays were performed immediately after estimation of protein concentration.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 31, 2023. ; https://doi.org/10.1101/2023.01.31.526483 doi: bioRxiv preprint DNA methyltransferase assay DNA methyltransferase (DNMT) activity of nuclear extracts from P. cynomolgi sporozoites, P. vivax sporozoites, and uninfected mosquito salivary glands were measured using the Epiquik 5 DNMT activity/inhibition assay ultra-kit (cat P-3010) following the manufacturer's instructions. Purified bacterial DNMT enzyme was used as a positive control. A blank control was used to subtract the residual background values. Each reaction was performed in duplicate. DNMT activity was measured in relative unit fluorescence per h per mg of protein for 10 min at 1 min intervals.

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Bisulfite conversion and library preparation P. cynomolgi and P. vivax sporozoites were lysed using 100 µl of lysis buffer containing 20mM Tris (pH8.1), 20 mM EDTA, 400 mM NaCl, 1 % SDS (w/v) for 30 min at room temperature followed by addition of 20 µl of proteinase K (20 mg/ml) to the pellet and incubated at 55 °C 15 overnight. The gDNA mixture was purified with phenol-chloroform followed by chloroform. Precipitation of gDNA was performed using chilled ethanol and treated with RNase A followed by another round of ethanol precipitation. 50 ng of unmethylated lambda DNA was added as a control to each sample before bisulfite conversion of the DNA. 500 ng of gDNA of each sample was used for the bisulfite conversion following the manufacturer's instructions (Epitect fast 20 bisulfite conversion kit, Qiagen cat59824). Libraries from bisulfite converted DNA were prepared using the Accel-NGS methyl-Seq DNA library kit (Swift biosciences cat 30024). Libraries were generated following the manufacturer's instructions and DNA was cleaned through SPRI select beads (Beckman Coulter). Libraries were sequenced using the NOVASeq platform.

DNA methylation analysis
Four sets of reads for P. vivax and P. cynomolgi were analyzed. Read qualities were checked with FastQC v0.11.8. FastQC indicated the presence of adapter contamination and overrepresented kmers. As a result, (1) the first 9-14 base pairs were trimmed and (2) reads with overrepresented k-30 mers were discarded (see fig. S9 for summary statistics after the cleaning step). Reads were mapped against the corresponding reference genomes downloaded from PlasmoDB (namely, PlasmoDB-48_Pfalciparum3D7, PlasmoDB-48_PcynomolgiB and PlasmoDB-48_PvivaxP01) using Bismark v0.22.2 with default parameters. To determine the bisulfite conversion rate, reads were also mapped against the lambda phage (see fig. S9 for the conversion rate). Alignment files 35 for the replicates were merged together using Samtools v1.9. Read methylation levels were obtained using Bismark v0.22.2 with default parameters (see fig. S9).
A cytosine in the genome was considered methylated if (1) the number of reads covering that cytosine was higher than a given threshold (10 for P. falciparum, 5 for P. vivax and 3 for P. 40 cynomolgi) and (2) the ratio of methylated reads over all reads covering a cytosine was higher than a given threshold (we chose 0.1 for this second threshold). Genome-wide cytosine density and methylated cytosine density in Fig. 3A and B were calculated in 1 kbp non-overlapping sliding windows using a custom script (available at https://github.com/salehsereshki/pyMalaria). The distribution of CG, CHG, CHH methylation in Fig. 3C and D were obtained by computing the 45 number of methylated cytosines in each context over all the methylated cytosines. For the methylation analyses in genes in Fig. 3E, (1) 500 bp flanking regions and gene body were split into five bins, (2) methylation levels were averaged across all the genes using a custom script . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 31, 2023. ; https://doi.org/10.1101/2023.01.31.526483 doi: bioRxiv preprint (available at the https://github.com/salehsereshki/pyMalaria). To study the correlation between cytosine methylation and gene expression, the same gene body computation was done for the 10% high and low expressed genes using a previously reported P. vivax transcriptome (20). These plots are represented in Fig. 3F.

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Additional ReFRAME hit confirmation using an improved P. vivax liver stage assay Twelve hits were re-confirmed using the 12-day radical cure assay, implementing three assay improvements (41). First, 100 μM 1-aminobenzotriazole (ABT, Caymen Chem cat 15252) was added to media on treatment days to reduce hepatic metabolism. Second, the assay endpoint was 10 extended 4 days to allow for nonviable liver stage forms to be cleared from cultures and therefore not be quantified during high-content imaging. Third, nigericin replaced monensin as the positive ionophore control. Confirmation was performed with one independent experiment for all compounds except cadralazine, which was confirmed in four independent experiments.

Epigenetic inhibitor library screen against P. vivax liver stages
The Epigenetic Inhibitor library (Targetmol, cat L1200), containing 773 compounds at 10 mM, was purchased and re-plated in pintool-ready 384-well source plates with 200 μM nigericin and DMSO control wells. The library was screened using the 12-day radical cure assay noted above.

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The 24 hits exhibiting the highest inhibition against hypnozoites were replated in a dose-response for confirmation of activity in a 12-day radical cure assay as described above. Confirmation was performed with two independent experiments.
Assessment of effect of ABT on hepatic cytochrome P450 3A4 activity 25 Two experiments were performed, one on uninduced PHHs, and another on rifampicin-induced PHHs (BioIVT, lot BGW). Cells were thawed and 18,000 live cells/well were seeded into collagen-coated 384-well plates as described above. Media was exchanged every-other-day until day 7 post-seed when media exchange included a dilution series of ABT. One hour after addition 30 of ABT, cytochrome P450 3A4 activity (CYP3A4) was measured using a luciferin-IPA kit (Promega cat V9001) following the lytic protocol with 3 μM IPA. Lysed well contents were transferred to a white 384-well luminometer plate (Greiner Bio-One cat 201106) before reading on a Spectramax i3X (Molecular Devices) with a 1 sec integration time. In the second experiment, cells were similarly seeded and cultured before addition of 25 μM rifampicin (MP Biomedial cat 35 BP2679-250), or an equivalent v/v DMSO vehicle control, in media on days 4 and 6. At day 7 post-seed, CYP3A4 activity was measured following addition of ABT as above. The fold change was calculated between induced and uninduced wells at each ABT dilution point.
Assessment of effect of ABT on hepatic metabolism using mass spectrometry 40 Primary human hepatocytes (lot BGW, BioIVT) were thawed and 18,000 live cells/well were seeded into collagen-coated 384-well plates as described above. Media was exchanged everyother-day until day 7 post-seed when cells were treated with 100 μM ABT, or an equivalent v/v vehicle control, in media for 1 h. Cells were then incubated with standard substrates for 45 characterization of phase I and II hepatic metabolism, including: 30 μM 7-hydroxycoumarin (UGT/ST), 40 μM coumarin (CYP2A6), 500 μM chlorzoxazone (CYP2E1), 50 μM dextromethorphan (CYP2D6), 24 μM midazolam (CYP3A4/5), 500 μM S-mephenytoin . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 31, 2023. ; https://doi.org/10.1101/2023.01.31.526483 doi: bioRxiv preprint (CYP2C19), 600 μM testosterone (CYP3A4), 1 mM tolbutamide (CYP2C9), 500 μM phenacetin (CYP1A2), or 400 μM bupropion (CYP2B6). The reaction was stopped at 1 h by addition of an equal volume of ice cold methanol. Metabolite formation was quantified using UPLC-MS/MS or LC-MS/MS (7-HC, 7-HCS, and 7-HCG). Samples were thawed, vortexed, and centrifuged for 5 min at 5000 rpm. Standards, controls, blanks, and study samples were added to an HPLC 5 autosampler vial and injected into the UPLC-MS/MS or LC-MS/MS systems. Analyses were run using an Acquity UPLC (Waters) or Agilent 1100 HPLC (Agilent) and Quattro premier XE (Waters) or Quattro Premier ZSpray (Waters) mass spectrometers. Quantification was performed using a quadratic least squares regression algorithm with 1/X 2 weighting, based on the peak area ratio of substrate or metabolite to its internal standard. Metabolite formation rate was calculated 10 as pmol/min/10 6 cells.

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Because cadralazine was found substantially more potent against hypnozoites than hydralazine (Fig. 1B), it was selected for a rhesus macaque pharmacokinetic study in which plasma levels were measured over 24 h following an oral dose of 1 mg/kg, which was calculated to be well-tolerated, and 30 mg/kg, which was calculated to likely cause drug-induced hypotension. Plasma exposure 20 following the 30 mg/kg dose was found to cover for several hours the in vitro EC90, and without noticeable side effects, indicating further studies were warranted to assess in vivo efficacy (fig. S16).
Immunofluorescent staining of 5mC and 5hmC in P. vivax blood stage parasites 25 An immunofluorescent staining approach has been used to detect both 5mC and 5hmC in P. falciparum blood stage parasites (18), thus we sought to confirm these marks in P. vivax blood stages. P. vivax blood samples were collected between 2017 and 2019 by active and passive case detection from individuals residing in Mondolkiri, Eastern Cambodia. The presence of P. vivax 30 was determined using an RDT (CareStartTM Malaria Pf/pan RDTs, Accesbio) or microscopy and monoinfections were confirmed by qPCR using species-specific primers (55). Venous blood used was collected in lithium heparin tubes and immediately processed on-site in a mobile laboratory. Leukocytes were depleted using NWF filters (56). The leukocyte depleted parasitized red blood cells were cryopreserved using glycerolyte 57 solution (Baxter) and immediately stored in liquid 35 nitrogen (57). Blood isolates were thawed by addition of 12%, then 1.6%, and then 0.9% (w/v) NaCl solution followed by heparin treatment for 10 min at 37 °C. Blood stage parasites were then purified from thawed isolates using a KCl-Percoll density gradient (58) followed by a wash with RPMI and two washes with 1× PBS. Parasites were then fixed with 3% (v/v) paraformaldehyde and 0.01% (v/v) glutaraldehyde in 1× PBS for 1 h at 4 °C. After fixation, blood stage parasites 40 were permeabilized, denatured, neutralized, quenched, and blocked as described above. Staining for 5mC and 5hmC was carried out as described above except the primary and secondary antibodies were diluted to 1 μg/mL instead of 10 μg/mL. Parasites were stained with 10 μg/mL Hoechst 33342 for 30 min at room temperature and then washed twice with 1× PBS after staining. Parasites were mounted on a coverslip and imaged with a 100× objective on a Leica DM250.

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While we did detect 5mC and 5hmC methylation in residual human white blood cells, we could . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 31, 2023. ; https://doi.org/10.1101/2023.01.31.526483 doi: bioRxiv preprint not confirm positive 5mC or 5hmC staining in P. vivax blood stage parasites from these isolates ( fig. S17). These negative results could be due to one or more factors. First, while P. falciparum blood stage cultures can reach parasitemias above 10%, P. vivax blood stages cannot be propagated in vitro and the parasitemia of isolates is typically just above the level of detection. Second, P. vivax blood stage isolates were cryopreserved before staining and the stability of DNA methylation 5 after cryopreservation is unknown. Third, the hydrochloric acid treatment needed to denature chromatin during the stain protocol causes red cells to aggregate, thereby making finding and imaging P. vivax blood stages difficult.

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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 31, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023 Fig. S1. 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, 5 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 10 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.

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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 31, 2023. ; https://doi.org/10.1101/2023.01.31.526483 doi: bioRxiv preprint Fig. S2. 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 5 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 10 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.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 31, 2023. ; https://doi.org/10.1101/2023.01.31.526483 doi: bioRxiv preprint Fig. S3. 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. 5 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.

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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 31, 2023. ; https://doi.org/10.1101/2023.01.31.526483 doi: bioRxiv preprint  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 31, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023  . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 31, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023  . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 31, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023 Fig. S7. 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 5 quantified area of signal per form. (B) Histogram of growth area all parasites quantified for Case 1, 2, and 3 in Fig. 2. Hypnozoites were classified as forms with an area 125µm 2 and smaller.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 31, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023  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. Significance determined using Kurskal-Wallis tests for hypnozoites and schizonts, with Dunn's multiple 10 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 ≤ 240) or all schizonts (7 ≤ n ≤ 26) in culture, 2' indicates a secondary stain only control.

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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 31, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023  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 10 read sets, percentage of mapped reads, read methylation levels, conversion rate and genomewide methylation levels from bisulfite sequencing.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made   (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.

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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made   (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.

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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 31, 2023. ; https://doi.org/10.1101/2023.01.31.526483 doi: bioRxiv preprint Fig. S13. Characterization of primary human hepatocyte (PHH) metabolism following 1aminobenzotriazole (ABT) treatment. (A) PHH lot BGW were seeded into 384-well plates and cultured for 7 days before addition of a dilution series of ABT in media. Cytochrome P450 3A4 activity (CYP3A4) was measured using luciferin-IPA (Promega). RLU: relative luminescence 5 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 ABT in media. Fold change was calculated based on matching uninduced controls. Data are from one independent 10 experiment. (C) PHH lot BGW were seeded in 384-well plates and cultured for 7 days before treatment with 100 μM 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.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 31, 2023. ; https://doi.org/10.1101/2023.01.31.526483 doi: bioRxiv preprint Fig. S15. Epigenetic Inhibitor library screen and hits. (A) Index chart of an epigenetic inhibitor library screened against P. vivax hypnozoites in a v3 (12-day + ABT) assay. Teal circle: library, black square: DMSO, pink triangle: 200 nM nigericin. (B) Structures of epigenetic inhibitor library hits which were confirmed to be active against P. vivax hypnozoites in dose-5 response assays; blue: histone deacetylase inhibitors.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 31, 2023. ; https://doi.org/10.1101/2023.01.31.526483 doi: bioRxiv preprint Fig. S16. 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.

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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 31, 2023. ; https://doi.org/10. 1101/2023 Fig. S17. 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 5 10 µm. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 31, 2023. ; https://doi.org/10.1101/2023.01.31.526483 doi: bioRxiv preprint Data S1. (separate file) Details for chemistry (batches, suppliers, structures); individual run data used to calculate all pEC50s and pCC50s; details for biologicals; details for reagents and replication. 5 Data S2. (separate file) Raw pharmacokinetic data report from Wuxi for cadralazine in rhesus macaques.

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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 31, 2023. ; https://doi.org/10.1101/2023.01.31.526483 doi: bioRxiv preprint