Chemical biology approaches have shown that hypnozoites become insensitive to most legacy antimalarials after 5 days in culture, indicating they must mature following hepatocyte infection (Maher et al., 2021; Posfai et al., 2020). Hypnozoite maturation was also noted in recent single-cell transcriptomic analyses of P. vivax liver stages, which demonstrate distinct population clusters of maturing and quiescent hypnozoites (Mancio-Silva et al., 2022; Ruberto et al., 2022). Importantly, discovery and development of hit compounds with radical cure activity in vivo, which includes elimination of hypnozoites in the liver of malaria patients (Campo et al., 2015), requires screening against mature hypnozoites in vitro (Zeeman et al., 2016). While our 8 day P. vivax liver stage platform, in which sporozoites are infected into PHHs and then allowed to mature for 5 days before being treated with test compound (Maher, 2021), has been used for screening small libraries against mature hypnozoites (Maher et al., 2021), the size of the ReFRAME library (12,823 compounds tested at 10 μM) presented a logistical challenge. We anticipated that dozens of P. vivax cases, each with a unique genetic background, would be needed to produce the sporozoites required to screen the 40 microtiter plates containing the library. To preclude the complex process of regular international shipments of infected mosquitoes, the P. vivax liver stage platform was successfully adapted and set up in research labs in two distinct malaria endemic areas, the Shoklo Malaria Research Unit (SMRU) in Thailand and the Institute Pasteur of Cambodia (IPC). The screening library was divided between both sites to enable concurrent progress; ultimately, 36 P. vivax cases from either site were needed to complete the primary screen over the course of 18 months (Figure 1A, Figure 1—figure supplement 1, Supplementary file 1).

We selected 72 compounds for confirmation of activity against hypnozoites in a dose–response format. These compounds were counter-screened for additional antimalarial activity against P. falciparum blood stages and P. berghei liver schizonts and tested for cytotoxicity against HEK293T and HepG2 human cell lines (Table 1). Following confirmation in dose–response assays, some hits exhibited moderate selectivity and potency, with pEC₅₀’s ranging from 5.42 to 7.07 (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) (Table 1). Colforsin daropate, rhodamine 123, and poziotinib are used to treat cancer and have known human targets, indicating that the targeted host pathways may be critical for hypnozoite persistence. As an example, poziotinib inhibits HER2, a tyrosine protein kinase associated with the downregulation of apoptosis and metastasis (Kavarthapu et al., 2021). We recently reported that host apoptotic pathways are downregulated in P. vivax-infected hepatocytes (Ruberto et al., 2022). Poziotinib could therefore act by upregulating apoptotic pathways in infected host cells. MS-0735, an analog of our previously reported hypnozonticidal hit, MMV018983 (Maher et al., 2021), is a ribonucleotide-reductase (RNR) inhibitor and used as an antiviral. The apparent need for nonreplicating hypnozoites to produce deoxyribonucleosides for DNA synthesis is peculiar. However, it has been reported that RNR is also critical for DNA damage repair (Elledge et al., 1992), is important for maintaining cancer cell dormancy (Evans and Lin, 2015), and is expressed in P. vivax liver schizonts and hypnozoites (Ruberto et al., 2022). We also rediscovered previously reported hypnozonticidal compounds included in the library, including the ionophore narasin (Maher et al., 2021) and the 8-aminoquinoline plasmocid (Schmidt and Schmidt, 1949; Figure 1—figure supplement 1, Table 1).

From our analysis of primary screen activity, we noted several hydrazinophthalazine vasodilators were potentially active (Figure 1—figure supplement 1) and selected 10 hydrazinophthalazine analogs for dose–response confirmation and counterscreen assays. Three hydrazinophthalazines analogs – cadralazine, pildralazine, and hydralazine – were active against mature hypnozoites, with cadralazine displaying the best combination of potency (pEC₅₀ = 6.33 ± 0.33), maximal inhibition near 100%, and selectivity over PHH (>21-fold), HEK293T (>85-fold), and HepG2 (>79-fold) cells (Figure 1B, Table 1). Hydralazine, which was FDA-approved in 1953, is currently one of the world’s most-prescribed antihypertensives, and on the WHO list of essential medicines (World Health Organization, 2019). Cadralazine, which was developed in the 1980s as an improvement over hydralazine, was abandoned due to side effects and only licensed in Italy and Japan (McTavish et al., 1990). Hydrazinophthalazines have been shown to inhibit human DNA methyltransferases (DNMT) (Cornacchia et al., 1988; Singh et al., 2009) and hydralazine has also been recently used to study potential DNA methylation patterns in the P. falciparum asexual blood stages (Ponts et al., 2013). Similar to our previous report (Ponts et al., 2013), these hydrazinophthalazines were inactive when tested against P. berghei liver schizonts, P. cynomolgi asexual blood stages, and P. falciparum asexual blood stages (Table 1—source data 1), suggesting that hypnozoite quiescence may be biologically distinct from developing schizonts (Maher et al., 2021). While hydrazinophthalazines may act on infected hepatocytes and not directly on the parasite, their distinct selectivity suggests that their effect is likely on a host or parasite pathways and not simply due to cytotoxicity in the host cell. Hydralazine and cadralazine were not identified as potential hits in any of the 112 bioassay screens of the ReFRAME published to date (Su, 2024), suggesting these compounds specifically target P. vivax liver stages and not promiscuously active compounds.

Methods for the robust culture of P. vivax hypnozoites were only recently reported, leading to several new reports on hypnozoite biology and radical cure drug discovery (Roth et al., 2018, Gural et al., 2018). Consequentially, some hypnozoite-specific discoveries appear to be platform-specific (Mancio-Silva et al., 2022; Ruberto et al., 2022). Select hits were shared with the Novartis Institute for Tropical Diseases (NITD), where the hypnozonticidal activity and potency of cadralazine (pEC₅₀ = 6.09 ± 0.45), hydralazine (pEC₅₀ = 6.20), and poziotinib (pEC₅₀ = 6.17) were independently confirmed in a similar 8-day P. vivax screening platform using a P. vivax case from southern Thailand (Figure 1B, Figure 1—figure supplement 2). Independent confirmation of these hits indicates their activities are not merely platform-specific and are, rather, more broadly descriptive of hypnozoite chemo-sensitivity.

Following our screening and hit confirmation, we investigated the potency, in vivo stability, and tolerability profile of our confirmed hits and chose cadralazine and hydralazine for repurposing as radical cure antimalarials. Currently, the gold-standard model for preclinical assessment of in vivo anti-relapse efficacy is rhesus macaques infected with Plasmodium cynomolgi, a zoonotic, relapsing species closely related to P. vivax (Joyner et al., 2015). Because we found cadralazine substantially more potent against hypnozoites than hydralazine, it was selected for a rhesus macaque pharmacokinetic study in which plasma levels were measured over 24 hr 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 (Hauffe and Dubois, 1984; Leonetti et al., 1988; Bonardi et al., 1983). The 30 mg/kg dose resulted in maximum plasma concentration of 13.7 μg/ml (or 48.2 μM) and half-life of 2.19 ± 0.24 hr, which was sufficient to cover the in vitro EC₉₀ for several hours without noticeable side effects (Figure 1—figure supplement 3). As another prerequisite for in vivo validation, we next sought to confirm and measure the potency of cadralazine and other ReFRAME hits against P. cynomolgi B strain hypnozoites in vitro using an 8-day assay featuring primary simian hepatocytes (PSH) at NITD. While poziotinib was active against P. cynomolgi hypnozoites when tested in two of three different PSH donor lots (pEC₅₀ = 5.67 and 5.95) (Supplementary file 2) hydralazine and cadralazine were found inactive when tested in all three different PSH donor lots (Figure 1B, Supplementary file 2). This negative result was later confirmed in an 8-day, simianized version of the platform at the University of Georgia (UGA) using the P. cynomolgi Rossan strain infected into two different PSH lots (Figure 1B). Altogether, these data highlight potential differences between P. vivax and P. cynomolgi and challenge the gold-standard model for preclinical assessment of in vivo anti-relapse efficacy in rhesus macaques.

As molecular tools to validate drug target in P. vivax are limited, we further interrogated the possible mechanism of action of hydrazinophthalazines using drug combination studies to assess synergy, additivity, or antagonism (Summers et al., 2022). We used 5-azacytidine, a known DNMT inhibitor (Christman, 2002), to investigate its effects on cadralazine treatment. When tested alone in dose–response from 50 μM, 5-azacytidine had no effect on hypnozoites. However, when added to cadralazine in fixed ratio combinations ranging from 8:1 to 1:8, 5-azacytidine increased the potency of cadralazine by ~2-fold across several combinations in two independent experiments (Figure 2, Figure 2—figure supplement 1). The most potent effect was detected using a 2:1 fixed ratio of cadralazine:5-azacytidine, resulting in an equivalent EC₅₀ decrease from 470 to 216 nM.

To further investigate if cadralazine could interact with P. vivax target(s), we aimed to detect and quantify DNA methylation in the P. vivax and P. cynomolgi genomes. Previous studies had identified the presence of low-level 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), and 5hmC-like marks throughout the P. falciparum genome (Ponts et al., 2013; Lucky et al., 2023; Hammam et al., 2020; Lenz et al., 2024). We first conducted an immunofluorescence staining assay using commercially available anti-5mC and anti-5hmC monoclonal antibodies to identify evidence of DNA methylation in P. vivax liver stages at 6 days post-infection. We found clear evidence of 5mC, but not 5hmC, in both schizonts and hypnozoites, morphologically consistent with the presence of 5mC in the parasite’s nucleus (Figure 3A, Figure 3—figure supplements 1–3). To segregate signals coming from the host hepatic nuclei, we used automated high-content imaging analysis on hundreds of individual P. vivax liver stage parasites as an unbiased approach for quantifying 5mC signal within parasites. Image masks were generated to quantify the area of 5mC or 5hmC stain within each parasite (Figure 3—figure supplement 4). The values were then plotted as stain area per hypnozoite or per schizont (Figure 3B). While some evidence of 5hmC-positive forms did appear from this analysis, the net 5hmC area per parasite was found significantly lower when compared to 5mC signals (Kruskal–Wallis tests, for hypnozoites H(7) = 194.3, p < 0.0001, for schizonts H(7) = 88.66, p < 0.0001). Similar results on the ratio of 5hmC to 5mC were also recently reported in P. falciparum blood stages (Lenz et al., 2024), confirming that 5mC marks are the predominant DNA methylation marks in both species.

Given the different susceptibility of P. cynomolgi hypnozoites to hydrazinophthalazines as compared to P. vivax, we performed automated high-content analysis of 5mC- and 5hmC-stained P. cynomolgi M/B-strain liver schizonts and hypnozoites at 8 and 12 days post-infection. Like P. vivax, we found both P. cynomolgi liver schizonts and hypnozoites are positive for 5mC, but not 5hmC. However, the 5mC stain morphology and intensity were relatively lower in P. cynomolgi hypnozoites versus P. vivax hypnozoites, suggesting potential divergence of DNA methylation pathways in these two species (Figure 3—figure supplement 5).

We next sought to confirm the presence of cytosine methylation in the P. vivax and P. cynomolgi genomes using mass spectrometry and bisulfite sequencing. We initially assessed that without an available single-cell sequencing approach, sequencing coverage of the parasite’s genome would be overwhelmed by the genomic material from the host cell as well as neighboring uninfected hepatocytes (Ruberto et al., 2022). We therefore collected sufficient genomic material from P. vivax and P. cynomolgi sporozoites to analyze the nucleoside mixture arising from the enzymatic digestion of genomic DNA by liquid chromatography–tandem mass spectrometry as well as for detection of DNMT activity using a commercial in vitro DNA methylation assay (Ponts et al., 2013). While we detected 5mC and DNMT activity in Plasmodium-enriched samples with these approaches, possible contamination by the mosquito’s microbiota could not be excluded (Figure 4—figure supplement 1). We next analyzed DNA methylation loci at single-nucleotide resolution using bisulfite sequencing of 3 × 10⁷ P. vivax sporozoites, generated from three different cases, as well as 3 × 10⁷ P. cynomolgi sporozoites (Figure 4A, B). A total of 161 and 147 million high-quality reads were sequenced for P. vivax and P. cynomolgi samples, respectively (Supplementary file 3). The average 5mC level detected across all cytosines was 0.49% and 0.39% for P. vivax and P. cynomolgi, respectively. These percentages are comparable to the 0.58% methylation level detected in P. falciparum blood stages (Ponts et al., 2013), but likely underestimate methylated loci considering the coverage we achieved (see methods).

We then monitored the distribution of detected 5mC along the P. vivax and P. cynomolgi chromosomes (Figure 4A, Figure 4—figure supplements 2 and 3) and observed a stable methylation level throughout the genomes, including in telomeric and sub-telomeric regions. We further examined the context of genome-wide methylations and, similar to what we previously observed in P. falciparum (Ponts et al., 2013), methylation was detected as asymmetrical, with CHH (where H can be any nucleotide but G) at 69.5% and 70.5%, CG at 16% and 15.7%, and CHG at 14.3% and 13.64%, for P. vivax and P. cynomolgi, respectively (Figure 4C). We then measured the proportion of 5mC in the various compartments of gene bodies (exons, the introns, promoters, and terminators) as well as strand specificity (Figure 4D, E). We observed a slightly increased distribution of 5mC in promoters and exons compared to the intronic region, as well as in the template versus non-template strand, in P. vivax and P. cynomolgi. These results were consistent with previous data obtained in P. falciparum and in plants (Ponts et al., 2013; Lucky et al., 2023). Such a strand specificity of DNA methylation patterns can affect the affinity of the RNA polymerase II and impact transcription; thus, we compared methylation levels to previously reported transcriptomic data from P. vivax sporozoites (Muller et al., 2019). The 5mC levels in 5′ flanking regions, gene bodies, and 3′ flanking regions were placed into five bins and compared to mRNA abundance, revealing an inverse relationship between methylation and mRNA abundance in the proximal promoter regions and the beginning of the gene bodies, with highly expressed genes appearing hypomethylated and weakly expressed genes hypermethylated (Figure 4F). These results suggest 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 malaria parasites. While these data will need to be further validated and linked to hypnozoite formation at a single-cell level, we have determined that 5mC is present at a low level in P. vivax and P. cynomolgi sporozoites and could control liver stage development and hypnozoite quiescence.

The success of the original screening platform protocol and secondary confirmation of several of our initial hits provided us an invaluable opportunity to develop an improved radical cure screening assay. The current iterations of our screening platform rely on high-content analysis of parasitophorous vacuole staining of the forms that persist up to the assay endpoint (Roth et al., 2018; Schafer et al., 2018). During the course of the ReFRAME primary screen, we found the day 8 endpoint was sufficient for some hit compounds to act. However, other compounds like the 8-aminoquinolines exhibit a ‘delayed death’ phenotype, which leads to a false-negative result (Maher et al., 2021). We therefore extended the assay by 4 days to allow attenuated forms to be cleared from the culture (Maher et al., 2021). Also, as our screening assays were performed with multiple lots of PHH and PSH, we detected some lot-specific results, possibly due to compound instability in the presence of hepatic metabolism (Figure 5—figure supplement 1). We therefore tested the metabolism inhibitor 1-aminobenzotriazole (1-ABT) in culture media to minimize the effect of lot-specific hepatic metabolism (Ortiz de Montellano and Mathews, 1981). We used a cytochrome P450 functional assay specific to CYP3A4 and determined that 100 μM of 1-ABT was sufficient to completely reduce CYP3A4 activity in both basal and rifampicin-induced PHH (Figure 5—figure supplement 1). This effect was further confirmed and quantified by mass spectrometry after 1 hr of treatment at 100 μM 1-ABT. We not only detected a 75% decrease in CYP3A4 activity, but also a more than 60% reduction of CYP2B6 and CYP2E1 activity along with lesser effects on CYP2C9, CYP1A2, and CYP2D6 (Figure 5). These changes were incorporated into our original 8-day protocol to design an improved 12-day assay (Maher, 2021) that we then validated by re-testing 12 ReFRAME hits. The modified assay did not drastically affect the potency of most hits (Figure 5—figure supplement 3), but helped resolve the hypnozonticidal activity of poziotinib (pEC₅₀ = 6.05), which had been previously confirmed in P. vivax and P. cynomolgi assays performed at NITD (Figure 5—figure supplement 2, Supplementary file 2, Figure 1—figure supplement 2). This assay was then used in all follow-up experiments.

To further confirm the importance of epigenetics in hypnozoite biology (Dembélé et al., 2014), we obtained a commercially available library containing 773 compounds targeting various inhibitors of epigenetic enzymes or pathways. These compounds were tested at 10 μM against P. vivax liver stages at both SMRU and IPC sites. We confirmed our initial hits in dose–response assays resulting in selective hypnozonticidal potency for 11 compounds targeting five different epigenetic mechanisms (Table 2). This includes the histone deacetylase inhibitors panobinostat (pEC₅₀ = 6.98 ± 0.18), AR42 (pEC₅₀ = 6.11 ± 0.24), abexinostat (pEC₅₀ = 5.48 ± 0.00), givinostat (pEC₅₀ = 5.35 ± 0.45), practinostat (pEC₅₀ = 5.32 ± 0.13), and raddeanin A (pEC₅₀ = 5.95 ± 0.00). Histone methyltransferase inhibitor hits included MI2 (pEC₅₀ = 5.48 ± 0.00), a compound that targets the interaction between menin (a global regulator of gene expression), and MLL (a DNA-binding protein that methylates histone H3 lysine 4 Cierpicki and Grembecka, 2014), and cyproheptadine (pEC₅₀ = 5.24 ± 0.34), which targets the SET-domain-containing lysine methyltransferase (Hirano et al., 2018). These results corroborate our hypothesis that epigenetic pathways regulate hypnozoites (Dembélé et al., 2014; Muller et al., 2019). Other hits, including 666-15 (pEC₅₀ = 5.88 ± 0.12), an inhibitor of the transcription factor cAMP response element-binding protein (Xie et al., 2015), and cerdulatinib (pEC₅₀ = 5.33 ± 0.20), a kinase inhibitor, suggest that signaling pathways may also be important for quiescence (Glennon et al., 2023).

Having identified several histone deacetylase inhibitors as directly or indirectly active on hypnozoites, we next screened compounds previously reported as inhibitors of P. falciparum acetyl-CoA synthetase (ACS), with downstream effects on histone acetylation (Summers et al., 2022). We found that one compound, MMV019721, was selectively active on mature P. vivax hypnozoites (Table 2). Given the evidence, MMV019721 is directly targeting P. falciparum ACS (Summers et al., 2022), this result suggests ACS also is a hypnozonticidal drug target. While the molecular techniques needed to confirm the direct interaction of MMV019721 and ACS in P. vivax are currently underdeveloped, our data supplement recent reports describing epigenetics as important regulators in P. vivax and P. cynomolgi at different stages of the parasite life cycle (Ruberto et al., 2022; Muller et al., 2019; Toenhake et al., 2023).

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The ReFRAME library was curated by assembling a list of developmental and FDA-approved chemistry from three databases (GVK Excelra GoStar, Clarivate Integrity, and Citeline Pharmaprojects). The original library consisted of 36 384-well plates (ReF01-ReF36, Supplementary file 1) containing 11,871 test compounds (Janes et al., 2018). While the original library was being screened, an additional set of four 384-well plates (ReF38–ReF41, Supplementary file 1) was added to the library, totaling 12,823 test compounds (Su, 2024). Source plates were made from the master library at Calibr at Scripps Research such that 3–5 μl of 10 mM solution was added to each well of a sterile, conical-bottom 384-well plate (Greiner Bio-One cat 784261). Most compounds were diluted in DMSO; however, a subset was diluted in water due to limited DMSO solubility. Plates were sealed and shipped on dry ice to SMRU and IPC and stored at –20°C prior to use. Column 24 of each plate was filled with 5 μl DMSO to serve as negative control wells. Control compounds included 1 mM monensin (positive control for hypnozoite and schizont activity), 1 mM the phosphatidylinositol 4-kinase inhibitor (PI4Ki) KDU691 or MMV390048 (positive control for schizont activity), 1 mM atovaquone (negative control for radical cure activity) and 10 mM tafenoquine (clinically relevant control for hypnozoite activity) (Roth et al., 2018; Maher et al., 2021).

The Thai human subjects protocols for this study were approved by the Institutional Ethics Committee of the Thai Ministry of Public Health and the Oxford Tropical Medicine Ethical Committee (TMEC 14-016 and OxTREC 40-14). The Cambodian human subjects protocols for this study were approved by the Cambodian National Ethics Committee for Health Research (100NECHR, 104NHECR, 111NECHR, 113NHECR, and 237NHECR). Protocols conformed to the Helsinki Declaration on Ethical Principles for Medical Research Involving Human Subjects (World medical association general assembly, 2004) and informed written consent was obtained for all volunteers or legal guardians. P. cynomolgi sporozoites were generated at Emory National Primate Research Center (ENPRC) using procedures approved by the Emory University Institutional Animal Care and Use Committee (PROTO201900110), as well as at UGA using procedures approved by UGA’s Institutional Animal Care and Use Committee (A2020 03-002-Y3-A15). P. cynomolgi sporozoites were also produced at the Armed Forces Research Institute of Medical Science under an IACUC-approved animal use protocol in an AAALAC International-accredited facility with a Public Health Services Animal Welfare Assurance and in compliance with the Animal Welfare Act and other federal statutes and regulations relating to laboratory animals (22-10). P. berghei sporozoites were generated by the Sporocore at UGA using procedures approved by UGA’s Institutional Animal Care and Use Committee (A2016 06-010-Y1-A0 and A2020 01-013-Y2-A3). Pharmacokinetic studies were conducted at WuXi AppTec Co, Ltd, in accordance with the WuXi IACUC standard animal procedures along with the IACUC guidelines that are in compliance with the Animal Welfare Act (National research council committee, 2011).

The complete, step-by-step protocol for the P. vivax liver stage assay is published (Maher, 2021). In summary, 2 days after assay plates (Greiner Bio-One cat 781956) were seeded with PHH, sporozoites were dissected from mosquito salivary glands and allowed to infect cultures. The 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 (Roth et al., 2018; Maher et al., 2021). On treatment days, a pintool was used to transfer 40 nl of compounds from the source plates into 40 μl of media in the assay plates, resulting in a 1000-fold dilution of all compounds. A single PHH lot, UBV, was first used for screening; however, once all available cryovials were used, screening was completed with lot BGW (Supplementary file 1). Screening was 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 (Supplementary file 1). Quantification of parasite growth was performed by fixing and staining cultures with recombinant mouse-anti P. vivax Upregulated in Infectious Sporozoites 4 (rPvUIS4) (Schafer et al., 2018), 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 μm² growth area.

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 (Figure 5—figure supplement 1), data were normalized using the Robust Z-score 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 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 protocol (Figure 1B, Table 1).

P. cynomolgi assays at UGA were performed using the step-by-step protocol for the P. vivax liver stage assay (Maher, 2021) with a few modifications. A Japanese macaque (Macaca fuscata) was intravenously infected with P. cynomolgi Rossan strain cryopreserved ring stage parasites (Collins et al., 2009) and allowed to reach patency. When parasitemia reached approximately 5000 parasites per µl, An. dirus mosquitoes were fed directly on the infected animal over a period of 3–4 days. The blood-fed mosquitoes were then checked for infection 6–8 days by dissecting and 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 compounds from the source plates to the assay plates. Quantification of P. cynomolgi liver stage growth was performed by fixing and staining cultures with 100 ng/ml mouse monoclonal antibody 13.3 (anti-GAPDH) obtained from The European Malaria Reagent Repository (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 μm² growth area.

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 Z99029) with the addition of 1% penicillin–streptomycin–neomycin (PSN) mix (Gibco cat 15640055) and 0.1% gentamicin in the case of P. vivax. Two days post-seeding, the hepatocytes 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 min at 200 × g. Once the sporozoites were removed after 24 hr 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 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 hr 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 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 (Mikolajczak et al., 2015). 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, 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 hr 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).

The SYBR Green I-based parasite proliferation assay (Plouffe et al., 2016) 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 (Ekland et al., 2011). Briefly, acoustic compound transfer (Labcyte Echo 550) was used to prepare assay-ready plates to which parasites in assay medium were added and incubated with compounds for 72 hr. 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 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 (13 point, 1:3 dilution series) were fitted with the four parameter Hill Equation.

Budralazine, cadralazine, and hydralazine (same catalog and batches as above) were tested using the [³H]-hypoxanthine drug susceptibility assay as previously described, with some modifications (Hott et al., 2015). Strain W2 (Oduola et al., 1988; Canfield et al., 1995) 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 mixture and incubated at 37°C. Cultures were sorbitol synchronized to achieve >70% ring stage parasites (Lambros and Vanderberg, 1979). Assays 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-mentioned gas mixture at 37°C for 48 hr; then, ³H-hypoxanthine (185 MBq, PerkinElmer cat NET177005MC) was added, and plates were incubated for another 24 hr. After 72 hr 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) representing relative parasite growth. Values were normalized to controls and plotted using CDD Vault. Potency values represent means of at least two independent experiments.

Budralazine, cadralazine, and hydralazine (same catalog and batches as above) were tested against P. cynomolgi DC strain using the [³H]-hypoxanthine drug susceptibility assay as previously described, with some modifications (Hott et al., 2015). 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 (2 g/l), and 5% washed rhesus erythrocytes. Cultures 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 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. ³H-hypoxanthine (185 MBq, PerkinElmer cat NET177005MC) was then added to all wells and plates were incubated under the previously mentioned conditions for 72 hr. After 72 hr the assay plates were 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.

For P. berghei liver stage assays, a colony of An. stephensi mosquitoes was maintained in the UGA Sporocore using methods previously described (Pathak et al., 2022). In summary, adults were fed 5% dextrose (wt/vol) and 0.05% para-aminobenzoic acid (wt/vol) soaked into cotton pads and kept at a temperature of 27°C, relative humidity of 75–85%, and a 12 hr light/dark cycle. PbGFP-LUC_CON sporozoites were produced as previously described (Pathak et al., 2022). In summary, female C57BL/6 or Hsd:ICR(CD-1) mice (Envigo) were injected intraperitoneally with 5 × 10⁶ to 5 × 10⁷ 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 (vol/vol, 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 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 (Swann et al., 2016). 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 hr, and intracellular parasite growth was measured using bioluminescence. 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 (13 point, 1:3 dilution series) were fitted with the four parameter Hill Equation.

HepG2 (ATCC cat HB-8065, RRID:CVCL_0027) and HEK293T (ATCC cat CRL-3216, RRID:CVCL_0063) mammalian cell lines were maintained in Dulbecco’s modified Eagle medium (DMEM, Gibco) with 10% heat-inactivated HyClone FBS (GE Healthcare Life Sciences), 100 IU penicillin, and 100 µg/ml streptomycin (Gibco) at 37°C with 5% CO₂ in a humidified tissue culture incubator. Cultures were routinely confirmed free of mycoplasma via Mycoalert (Lonza) using the manufacturer’s protocol. To assay mammalian toxicity of hit compounds, 750 HepG2 and 375 HEK293T cells/well were seeded, respectively, in assay media (DMEM, 2% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin) in 1536-well, white, tissue culture-treated, solid bottom plates (Corning cat 9006BC) that contained acoustically transferred compounds in a threefold serial dilution starting at 40 µM. After a 72-hr incubation, 2 µl of 50% Cell-Titer Glo (Promega cat G7573) diluted in water was added to the cells and luminescence measured on an EnVision Plate Reader (PerkinElmer).

Powders of cadralazine (same batch as above), 5-azacytidine (Caymen Chem, cat 11164), and nigericin were diluted to 50 mM, 50 mM, and 200 μM, respectively, in DMSO, before being diluted to 100 μM, 100 μM, and 400 nM, respectively, in hepatocyte culture media (BioIVT, cat Z99029). Cadralazine and 5-azacytidine were then plated in the first column of two 96-well plates at volumetric 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 such that the net volume per well was 200 μl (nigericin and DMSO controls were also diluted as such). Each mixture was then diluted in a 12-point, twofold dilution series by mixing 100 μl of mixture to 100 μl media in subsequent columns using a multichannel pipettor. A 384-well P. vivax liver stage assay plate was started using the 12-day protocol as above, and on day 5, 6, and 7 post-infection, media was removed from the 384-well plate using the inverted spin method Maher, 2021 followed by addition of 20 μl of fresh media. Then, a multichannel pipettor was used to transfer 20 μl of the mixtures (made fresh daily) from the 96-well dilution series plates to the 384-well plates, thereby establishing a highest 1:0 and 0:1 treatment dose of 50 μM. The assay was fixed, stained, imaged, and parasite growth quantified as described above. Parasite growth data were normalized to the DMSO control and loaded into Prism (GraphPad) for curve fitting using the setting ‘log(inhibitor) vs. response – variable slope (four parameters) least squares fit’. The EC₅₀’s of each ratio were used to calculate Fractional Inhibitory Concentrations (FICs) and plot isobolograms as previously described (Ohrt et al., 2002).

Sporozoites from three different P. vivax cases were infected into PHH lot BGW at day 2 post-seed (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 above. Cultures were fixed at day 6 post-infection and stained with rPvUIS4 and Hoechst 33342 as previously described (Maher, 2021; Schafer et al., 2018). Cultures were then stained with either rabbit anti-5mC monoclonal antibody (clone RM231, Thermo Fisher Scientific cat MA5-24694, RRID:AB_2665309) or rabbit anti-5hmC monoclonal antibody (clone RM236, Thermo Fisher Scientific cat MA5-24695, RRID:AB_2665308) using methods adapted from those previously described by Hammam et al., 2020. In summary, cultures were re-permeabilized with 0.1% (vol/vol) 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 and washed thrice with 1× PBS. Cultures were then blocked with 0.1% (vol/vol) Tween 20 and 2% (wt/vol) bovine serum albumin for 10 min at room temperature and washed thrice with PBS. 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 (Thermo Fisher 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 was prepared as above and stained with secondary antibody only (2’ control, Figure 3B). High-resolution images of individual parasites and PHH nuclei were obtained by capturing eight planes in the Z dimension using a 100× objective on Deltavision Core (GE Healthcare Life Sciences) and deconvoluted using softWoRx (GE Healthcare Life Sciences) (Figure 3A, Figure 3—figure supplements 1–3). An ImageXpress Micro high-content imager was used to quantify methyl-cytosine modifications for the entire population of parasites from each case. 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 parasite was used to define parasite objects, and the 5mC or 5hmC staining of host cell nuclei was used to define positive methyl-cytosine modification objects. The two-dimensional area of intersection of both objects was then quantified for each parasite, and forms less than 125 μm² were quantified as hypnozoites (Figure 3—figure supplement 4).

Japanese macaques (M. fuscata) were intravenously infected with P. cynomolgi M/B strain (Joyner et al., 2019) and allowed to reach patency before skin feeding to An. dirus mosquitoes as described above. One round of macaque infection, mosquito dissection, and culture infection was performed with PSH lot NPI, and a second round was performed with PSH lot NNF. Two days after assay plates (Greiner Bio-One cat 781956) were seeded with 20,000 PSH per well, sporozoites were dissected from mosquito salivary glands at day 16 post-bloodmeal and allowed to infect cultures. Cultures were fixed on day 8 (experiment 1) or 12 (experiment 2) post-infection and stained 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 the 5mC or 5hmC staining of host cell nuclei was used to define positive methyl-cytosine modification objects. The two-dimensional area of intersection of both objects was then quantified for each parasite, and forms less than 105 μm² were categorized as hypnozoites (Figure 3—figure supplement 5).

For quantification of 5mC modification levels by mass spectrometry, sporozoites from 3 different P. vivax cases, numbering 18.7 × 10⁶ from case 1, 101 × 10⁶ from case 2, and 14.7 × 10⁶ from case 3, were dissected from infected An. dirus mosquitoes at IPC as previously described (Maher, 2021) and cryopreserved as previously described (Singh et al., 2016). For quantification of DNMT activity from nuclear extracts, sporozoites from two different P. vivax cases, numbering 21 × 10⁶ from case 1 and 20 × 10⁶ 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 × 10⁶ sporozoites for mass spec and 34.1 × 10⁶ sporozoites for DNMT activity assays were also collected from An. dirus mosquitoes infected from feeding on a rhesus macaque infected with P. 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 dissected and cryopreserved. For mapping of methyl-cytosine modifications by bisulfite sequencing, sporozoites from three different P. vivax cases, numbering 9.8 × 10⁶ from case 1, 12.3 × 10⁶ from case 2, and 15.1 × 10⁶ from case 3, were dissected from infected An. dirus mosquitoes at IPC and cryopreserved as described above. A total of 5.3 × 10⁶ sporozoites were also collected from An. dirus mosquitoes infected from feeding on a rhesus macaque infected with P. 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.

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°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 14,500 RCF for 30 min at 4°C and the supernatant was 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 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 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, 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.

Cryopreserved sporozoites, or parasites extracted from red blood cells by saponin lysis, were resuspended in 1 ml of cytoplasmic lysis buffer (20 mM HEPES pH 7.9, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM 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 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 1× PBS. Nuclei pellets were resuspended in 100 µl of nuclei lysis buffer (20 mM HEPES pH 7.9, 0.1 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM 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 RCF. Protein concentration of nuclear extract was quantified by BCA assay and DNMT assays were performed immediately after estimation of protein concentration.

DNMT activity of nuclear extracts from P. cynomolgi sporozoites, P. vivax sporozoites, and uninfected mosquito salivary glands was measured using the Epiquik 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.

P. cynomolgi and P. vivax sporozoites were lysed using 100 µl of lysis buffer containing 20 mM Tris (pH 8.1), 20 mM EDTA, 400 mM NaCl, 1% SDS (wt/vol) 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 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 bisulfite conversion kit, QIAGEN cat 59824). 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.

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 k-mers. As a result, (1) the first 9–14 base pairs were trimmed and (2) reads with overrepresented k-mers were discarded (see Supplementary file 3 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 Supplementary file 3 for the conversion rate). Alignment files for the replicates were merged together using Samtools v1.9. Read methylation levels were obtained using Bismark v0.22.2 with default parameters (see Supplementary file 3).

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. 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 Figure 4A, B were calculated in 1 kbp non-overlapping sliding windows using a custom script (available at https://github.com/salehsereshki/pyMalaria copy archived at Sereshki, 2021). The distribution of CG, CHG, and CHH methylation in Figure 4C, D was obtained by computing the number of methylated cytosines in each context over all the methylated cytosines. For the methylation analyses in genes in Figure 4E, (1) 500 bp flanking regions and gene body were split into five bins and (2) methylation levels were averaged across all the genes using a custom script (available at the https://github.com/salehsereshki/pyMalaria; Sereshki, 2021). 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 (Muller et al., 2019). These plots are represented in Figure 4F.

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 1-ABT. One hour after addition of 1-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-s integration time. In the second experiment, cells were similarly seeded and cultured before addition of 25 μM rifampicin (MP Biomedial cat BP2679-250), or an equivalent vol/vol DMSO vehicle control, in media on days 4 and 6. At day 7 post-seed, CYP3A4 activity was measured following addition of 1-ABT as above. The fold change was calculated between induced and uninduced wells at each 1-ABT dilution point.

PHHs (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 every other day until day 7 post-seed when cells were treated with 100 μM 1-ABT, or an equivalent vol/vol vehicle control, in media for 1 hr. Cells were then incubated with standard substrates for 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 (CYP2C19), 600 μM testosterone (CYP3A4), 1 mM tolbutamide (CYP2C9), 500 μM phenacetin (CYP1A2), or 400 μM bupropion (CYP2B6). The reaction was stopped at 1 hr 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 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 /X² weighting, based on the peak area ratio of substrate or metabolite to its internal standard. Metabolite formation rate was calculated as pmol/min/10⁶ cells.

Twelve hits were re-confirmed using the 12-day radical cure assay, implementing three assay improvements Maher, 2021. First, 100 μM 1-ABT (Caymen Chem cat 15252) was added to media on treatment days to reduce hepatic metabolism. Second, the assay endpoint was 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.

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. 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. The ACS inhibitors MMV019721 and MMV084978 were kindly provided by MMV and tested in dose–response in a 12-day radical cure assay as described above. Potency was determined from four independent experiments.