1. Biochemistry and Chemical Biology
  2. Microbiology and Infectious Disease

A drug repurposing approach reveals targetable epigenetic pathways in Plasmodium vivax hypnozoites

  1. Steven P Maher  Is a corresponding author
  2. Malina A Bakowski
  3. Amélie Vantaux
  4. Erika L Flannery
  5. Chiara Andolina
  6. Mohit Gupta
  7. Yevgeniya Antonova-Koch
  8. Magdalena Argomaniz
  9. Monica Cabrera-Mora
  10. Brice Campo
  11. Alexander T Chao
  12. Arnab K Chatterjee
  13. Wayne T Cheng
  14. Vorada Chuenchob
  15. Caitlin A Cooper
  16. Karissa Cottier
  17. Mary R Galinski
  18. Anke Harupa-Chung
  19. Hana Ji
  20. Sean B Joseph
  21. Todd Lenz
  22. Stefano Lonardi
  23. Jessica Matheson
  24. Sebastian A Mikolajczak
  25. Timothy Moeller
  26. Agnes Orban
  27. Vivian Padín-Irizarry
  28. Kastin Pan
  29. Julie Péneau
  30. Jacques Prudhomme
  31. Camille Roesch
  32. Anthony Ruberto
  33. Saniya S Sabnis
  34. Celia L Saney
  35. Jetsumon Sattabongkot
  36. Saleh Sereshki
  37. Sangrawee Suriyakan
  38. Ratawan Ubalee
  39. Yinsheng Wang
  40. Praphan Wasisakun
  41. Jiekai Yin
  42. Jean Popovici
  43. Case W McNamara
  44. Chester Joyner
  45. François H Nosten
  46. Benoît Witkowski
  47. Karine G Le Roch
  48. Dennis E Kyle  Is a corresponding author
  1. Center for Tropical and Emerging Global Disease, University of Georgia, United States
  2. Calibr, a division of The Scripps Research Institute, United States
  3. Malaria Molecular Epidemiology Unit, Institute Pasteur of Cambodia, Cambodia
  4. Novartis Institute for Tropical Diseases, Novartis Institutes for Biomedical Research, United States
  5. Shoklo Malaria Research Unit, Mahidol-Oxford Tropical Medicine Research Unit, Thailand
  6. Department of Molecular, Cell, and Systems Biology, University of California, Riverside, United States
  7. Center for Vaccines and Immunology, College of Veterinary Medicine, University of Georgia, United States
  8. International Center for Malaria Research, Education and Development, Emory Vaccine Center, Emory National Primate Research Center, Emory University, United States
  9. Medicines for Malaria Venture (MMV), Switzerland
  10. BioIVT Inc, United States
  11. Division of Infectious Diseases, Department of Medicine, Emory University, United States
  12. Department of Computer Science and Engineering, University of California, Riverside, United States
  13. Department of Microbiology and Immunology, University of Otago, New Zealand
  14. School of Sciences, Clayton State University, United States
  15. Mahidol Vivax Research Unit, Mahidol University, Thailand
  16. Department of Entomology, Armed Forces Research Institute of Medical Sciences (AFRIMS), Thailand
  17. Department of Chemistry, University of California, Riverside, United States
  18. Environmental Toxicology Graduate Program, University of California, Riverside, United States
  19. Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, United Kingdom
https://doi.org/10.7554/eLife.98221.2
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Cite this article
  1. Steven P Maher
  2. Malina A Bakowski
  3. Amélie Vantaux
  4. Erika L Flannery
  5. Chiara Andolina
  6. Mohit Gupta
  7. Yevgeniya Antonova-Koch
  8. Magdalena Argomaniz
  9. Monica Cabrera-Mora
  10. Brice Campo
  11. Alexander T Chao
  12. Arnab K Chatterjee
  13. Wayne T Cheng
  14. Vorada Chuenchob
  15. Caitlin A Cooper
  16. Karissa Cottier
  17. Mary R Galinski
  18. Anke Harupa-Chung
  19. Hana Ji
  20. Sean B Joseph
  21. Todd Lenz
  22. Stefano Lonardi
  23. Jessica Matheson
  24. Sebastian A Mikolajczak
  25. Timothy Moeller
  26. Agnes Orban
  27. Vivian Padín-Irizarry
  28. Kastin Pan
  29. Julie Péneau
  30. Jacques Prudhomme
  31. Camille Roesch
  32. Anthony Ruberto
  33. Saniya S Sabnis
  34. Celia L Saney
  35. Jetsumon Sattabongkot
  36. Saleh Sereshki
  37. Sangrawee Suriyakan
  38. Ratawan Ubalee
  39. Yinsheng Wang
  40. Praphan Wasisakun
  41. Jiekai Yin
  42. Jean Popovici
  43. Case W McNamara
  44. Chester Joyner
  45. François H Nosten
  46. Benoît Witkowski
  47. Karine G Le Roch
  48. Dennis E Kyle
(2025)
A drug repurposing approach reveals targetable epigenetic pathways in Plasmodium vivax hypnozoites
eLife 13:RP98221.
https://doi.org/10.7554/eLife.98221.2
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eLife assessment

This paper reports a large drug repurposing screen based on an in vitro culture platform to identify compounds that can kill Plasmodium hypnozoites. This valuable work adds to the current repertoire of anti-hypnozoites agents and uncovers targetable epigenetic pathways to enhance our understanding of this mysterious stage of the Plasmodium life cycle. The data presented here are based on solid methodology and represent a starting point for further investigation of epigenetic inhibitors to treat P. vivax infection. This paper will be of interest to Plasmodium researchers and more broadly to readers in the fields of host-pathogen interactions and drug development.

https://doi.org/10.7554/eLife.98221.2.sa0

Abstract

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 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.

Introduction

Of the six species of Plasmodium that cause malaria in humans (Ansari et al., 2016), Plasmodium vivax is the most globally widespread (Howes et al., 2016). Vivax malaria now accounts for the most malaria episodes in countries with successful falciparum malaria control programs (Price et al., 2020). Controlling vivax malaria is complicated by the ability of P. vivax sporozoites, the infectious stage inoculated by mosquitoes, to invade hepatocytes and become quiescent (Wells et al., 2010; White et al., 2014). These quiescent ‘hypnozoites’ persist, undetectable, for months or even years before resuming growth and initiating a ‘relapse’ blood stage infection, leading to subsequent transmission back to mosquitoes (Adams and Mueller, 2017). New evidence suggests this transmission is expedited and silent as P. vivax liver merozoites can immediately form gametocytes instead of first having to establish an asexual stage blood infection, such as is the case for P. falciparum (Roth et al., 2018; Adapa et al., 2019; Schäfer et al., 2020; Mancio-Silva et al., 2022). Clinically, a compound with radical cure efficacy is one that removes all parasites from the patient, including hypnozoites in the liver (Campo et al., 2015).

Hypnozoites are refractory to all antimalarials except the 8-aminoquinolines, which were first identified over 70 years ago using low-throughput screening in avian malaria models (Rangel and Llinás, 2021). Primaquine was the first 8-aminoquinoline widely used for radical cure; however, efficacy is contingent on a large total dose administered in a 7- to 14-day regimen, leading to adherence problems and infrequent use in malaria control programs of endemic countries (Taylor et al., 2019). Tafenoquine–chloroquine was developed from primaquine as an improved single dose for radical cure (Llanos-Cuentas et al., 2019), but a recent clinical trial shows tafenoquine lacks efficacy when co-administered with the common antimalarial dihydroartemisinin-piperaquine, calling into question tafenoquine’s suitability in areas of high chloroquine resistance (Sutanto et al., 2023). Furthermore, 8-aminoquinolines cannot be administered to pregnant women or glucose-6-phosphate dehydrogenase-deficient individuals and are ineffective in persons with specific cytochrome P450 genotypes (Baird, 2019). For these reasons, the discovery and development of new chemical classes with radical cure activity are needed (Burrows et al., 2017).

Modern drug discovery typically relies on phenotypic screening and protein target identification (Schenone et al., 2013). For malaria, this approach ensures hits are acting on parasite targets and enables rational drug design, leading to several promising novel classes of antimalarials (Kuhen et al., 2014; Forte et al., 2021). However, due to lower cost and higher feasibility, current high-throughput screening for new antimalarials focuses almost entirely on blood or liver schizonts (Avery et al., 2014; Antonova-Koch et al., 2018). High-throughput antimalarial screening with a target chemo-profile for killing hypnozoites has only recently been made possible with the introduction of cell-based phenotypic screening platforms featuring a monolayer of hepatocytes infected with sporozoites, a portion of which go on to form hypnozoites (Valenciano et al., 2022). While the first hypnozonticidal hits from these platforms are just now being reported (Maher et al., 2021), protein target identification approaches for hypnozonticidal drug discovery are in their infancy as the transcriptome of hypnozoites has only recently been reported and robust methods for genetic manipulation of P. vivax are still underdeveloped (Ruberto et al., 2022; Bermúdez et al., 2018).

To address the lack of radical cure drug leads and targets, we used our advanced P. vivax liver stage platform to first screen the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) library (Janes et al., 2018). This library consists of approximately 12,000 developmental, approved, and discontinued drugs with the expectation that the repurposing of compounds with some optimization or regulatory success could expedite the decade-long path drugs typically progress through from discovery to licensure (Janes et al., 2018). To accomplish this screen, we assembled an international collaboration with laboratories in malaria-endemic countries whereby vivax-malaria patient blood was collected and fed to mosquitoes to produce sporozoites for infecting primary human hepatocytes (PHHs) in screening assays performed on-site. Interestingly, two structurally related compounds used for treating hypertension, hydralazine and cadralazine, were found effective at killing hypnozoites. Because these inhibitors have been shown to modulate DNA methylation (Cornacchia et al., 1988; Singh et al., 2009), we pursued and confirmed the existence of methyl-cytosine modifications in P. vivax sporozoite and liver stages. Having found in the ReFRAME screen a class of hits targeting an epigenetic pathway, we decided to confirm the importance of epigenetics in P. vivax hypnozoites and screened an additional commercial epigenetic inhibitor library using an improved version of our screening platform. Hypnozoites were found to be susceptible to several classes of epigenetic inhibitors, including six distinct histone deacetylase inhibitors and two inhibitors targeting histone methylation. To further assess the importance of histone acetylation in P. vivax liver stages, we tested inhibitors previously reported to be directly acting on P. falciparum acetyl-CoA synthetase, thereby modulating the pool of acetyl-CoA available for histone acetylation (Summers et al., 2022). We found MMV019721 selectively kills P. vivax and P. cynomolgi hypnozoites, implicating acetyl-CoA synthetase as an additional hypnozonticidal drug target. This work demonstrates that in lieu of traditional molecular biology methods, our screening platforms identify multiple, druggable epigenetic pathways in hypnozoites and add to the growing body of evidence that epigenetic features underpin biology in P. vivax and P. cynomolgi sporozoite and liver stages (Ruberto et al., 2022; Dembélé et al., 2014; Muller et al., 2019; Toenhake et al., 2023).

Results

ReFRAME library screening cascade, hit identification, and confirmation

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).

Figure 1 with 3 supplements see all
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Hypnozonticidal hit detection and confirmation.

(A) Index chart depicting the primary screen of the ReFRAME library against P. vivax hypnozoites in an 8-day assay. Hypnozoite counts were normalized by mean quantity per well for each plate (Z-score). Teal: library, black: DMSO, red: 1 μM monensin. (B) Dose–response curves for cadralazine against P. vivax and P. cynomolgi liver forms in 8-day assays at the IPC, UGA, and NITD. All replicate wells were plotted together from all independent experiments (n = 3 for P. vivax at IPC, n = 1 for P. vivax at NITD, n = 2 for P. cynomolgi at UGA, and n = 4 for P. cynomolgi at NITD), bars represent SEM.

Figure 1—source data 1

Source data for Figure 1 and supporting figures.

https://cdn.elifesciences.org/articles/98221/elife-98221-fig1-data1-v1.xlsx
Table 1
Dose–response confirmation and counterscreens of primary screen hits and analogs.

Primary screen hits and structurally or mechanistically related compounds were tested by dose–response in 8 day P. vivax liver stage assays at Institute Pasteur of Cambodia and counterscreened against P. berghei liver schizonts, P. falciparum asexual blood stages of strain Dd2 and W2, and human cell lines HEK293T and HepG2. Values represent pEC50 or pCC50 ± SD of all independent experiments (n = 2–6) for which a pEC50 or pCC50 was obtained. An asterisk (*) indicates only one independent experiment resulted in a calculated pEC50 or pCC50. pEC50 is the inverse log of potency in M concentration, e.g. pEC50 3 = 1 mM, pEC50 6 = 1 μM, and pEC50 9 = 1 nM.

CompoundStatusP. vivax hypnozoitesIPCP. vivax liver schizontsIPCPrimary human hepatocytesIPCP. berghei liver schizontsP. falciparum asexual blood stage, strain Dd2Cytotoxicity, HEK293TCytotoxicity, HepG2
(pEC50 ± SD)(pEC50 ± SD)(pCC50 ± SD)(pEC50 ± SD)(pEC50 ± SD)(pCC50 ± SD)(pCC50 ± SD)
Antihypertensives
 CadralazineRegistered6.33 ± 0.296.33 ± 0.18< 5.00< 5.00< 4.90< 4.404.43*
 PildralazineDiscontinued6.08 ± 0.27≤ 5.95< 5.00< 5.00< 4.90< 4.404.74*
 HydralazineRegistered5.75*5.42*< 5.00< 5.00< 4.90< 4.404.51*
 BudralazineRegistered< 5.00< 5.00< 5.005.88 ± 0.4< 4.90< 4.40< 4.40
 DihydralazinePreclinical< 5.00< 5.00< 5.005.53 ± 0.145.07 ± 0.074.7 ± 0.064.50 ± 0.11
 EndralazineDiscontinued< 5.00< 5.00< 5.00< 5.00< 4.904.51*4.47*
 MopidralazineDiscontinued< 5.00< 5.00< 5.00< 5.00< 4.90< 4.40< 4.40
 TodralazineUnknown< 5.00< 5.00< 5.00< 5.00< 4.90< 4.40< 4.40
 DramedilolPhase I< 5.00< 5.00< 5.00< 5.00< 4.904.73 ± 0.064.60 ± 0.06
 RGH-5526Phase I<< 5.00< 5.00< 5.00< 4.904.87 ± 0.194.67 ± 0.12
Anticancer
  Colforsin
  daropate		Registered7.07*< 5.00< 5.00< 5.00< 4.904.71 ± 0.174.41*
 Rhodamine 123Phase I5.23 ± 0.31≤ 5.48< 5.00< 5.005.28 ± 0.085.28 ± 0.34.65 ± 0.07
 PAN-811Phase II< 5.00< 5.00< 5.005.91 ± 0.295.66 ± 0.546.03 ± 0.235.77 ± 0.13
 PoziotinibPhase II< 5.00< 5.00< 5.005.23 ± 0.15.25 ± 0.035.27 ± 0.224.72 ± 0.16
Other
 NarasinAnimal use5.79 ± 0.26.50*< 5.009.09 ± 0.427.92 ± 0.137.57 ± 1.076.66 ± 0.58
 MS-0735Preclinical5.42*≤ 5.48< 5.006.22 ± 0.075.38 ± 0.096.07±0.226.05 ± 0.21
 PlasmocidDiscontinued≤ 5.48≤ 5.95< 5.005.70 ± 0.276.74 ± 0.564.96 ± 0.144.95 ± 0.37
Table 1—source data 1

Source data for Table 1.

https://cdn.elifesciences.org/articles/98221/elife-98221-table1-data1-v1.xlsx

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.

Synergy between cadralazine and 5-azacytidine

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.

Figure 2 with 1 supplement see all
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Synergistic effect of cadralazine and 5-azacytidine in P. vivax liver stage assays.

(A) Isobologram of cadralazine and 5-azacytidine activity against hypnozoites in fixed ratios of 1:0, 8:1, 6:1, 4:1, 2:1, 1:1, 1:2, 1:4, 1:6, 1:8, and 0:1, bars represent SD of FICs from two independent experiments. (B) Dose–response curves for cadralazine at the most synergistic fixed ratios (2:1, 4:1, and 8:1) against hypnozoites. Cadralazine alone is represented as 1:0, 5-azacytidine alone is represented as 0:1 and plotted on the cadralazine chart for comparison. Left and right charts represent two independent experiments, bars represent replicate wells at each dose.

Figure 2—source data 1

Source data for Figure 2 and supporting figures.

https://cdn.elifesciences.org/articles/98221/elife-98221-fig2-data1-v1.xlsx

Immunofluorescent detection of DNA methylation in P. vivax and P. cynomolgi liver stages

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.

Figure 3 with 5 supplements see all
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Cytosine modifications in P. vivax liver forms.

(A) Immunofluorescent imaging of a 5mC-positive (left) or 5hmC-negative (right) P. vivax hypnozoite (top) and schizont (bottom) at day 6 post-infection. White arrows indicate hepatocyte nuclei positive for 5mC or 5hmC. Bars represent 10 µm. (B) High-content quantification of 5mC or 5hmC stain area within hypnozoites or schizonts from sporozoites generated from three different P. vivax cases. Significance determined using Kruskal–Wallis tests, for hypnozoites H(7) = 194.3, p < 0.0001, for schizonts H(7) = 88.66, p < 0.0001, with Dunn’s multiple comparisons, *p < 0.05, ***p < 0.001, ****p < 0.0001, ns = not significant. Line, box, and whiskers represent median, upper and lower quartiles, and minimum-to-maximum values, respectively, of all hypnozoites (177 ≤ n ≤ 257) or all schizonts (30 ≤ n ≤ 142) in culture for each case, 2’ indicates a secondary stain only control.

Figure 3—source data 1

Source data for Figure 3 and supporting figures.

https://cdn.elifesciences.org/articles/98221/elife-98221-fig3-data1-v1.xlsx

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).

Detection of cytosine modifications in P. vivax and P. cynomolgi sporozoites using liquid chromatography–tandem mass spectrometry and bisulfite sequencing

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).

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Density of cytosine and methylated cytosine (5mC) in sporozoites.

(A) CG content of chromosome 1 for P. vivax and P. cynomolgi. The total number of cytosines was quantified on each strand using 1 kbp long non-overlapping windows. (B) The total number of methylated cytosines was quantified on each strand using 1 kbp long non-overlapping windows. (C) The number of 5mC present in all possible contexts (CG, CHG, and CHH) quantified throughout the genome of P. vivax and P. cynomolgi. (D) Repartitioned 5mC quantity within different compartments of the genome in P. vivax and P. cynomolgi. (E) Strand specificity of 5mC for all genes in P. vivax and P. cynomolgi. Flanking regions and gene bodies were divided into five bins, and the methylation level of each bin was averaged among all genes. Red: template strand, blue: non-template strand. (F) The previously reported mRNA abundance of P. vivax sporozoites was retrieved (Antonova-Koch et al., 2018) and genes ranked. The 5mC levels in 5′ flanking regions, gene bodies, and 3′ flanking regions were placed into five bins and are shown for highly expressed (90th percentile, left) and weakly expressed (10th percentile, right) genes. Red: template strand, blue: non-template strand.

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.

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Characterization of primary human hepatocyte (PHH) metabolism following 1-aminobenzotriazole (1-ABT) treatment.

PHH lot BGW was seeded in 384-well plates and cultured for 7 days before treatment with 100 μM 1-ABT for 1 hr, followed by addition of substrates for 1 hr and collection for analysis by mass spectrometry. Data are combined from two independent experiments, bars represent SD of all replicates. Significance determined by Student’s t tests, ****p < 0.0001, ***p < 0.001, **p < 0.01, ns, not significant.

Figure 5—source data 1

Source data for Figure 5 and supporting figures.

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Assay improvements and epigenetic inhibitor library screen

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).

Table 2
Additional epigenetic inhibitors with activity against P. vivax liver stages.
Epigenetic inhibitorTarget(s)Hypnozoite pEC50 ± SDLiver schizont pEC50 ± SDPHH nuclei pCC50 ± SD
PanobinostatHDAC6.98 ± 0.187.00 ± 0.155.68 ± 0.18
AR42HDAC6.11 ± 0.246.30 ± 0.205.29 ± 0.27
Raddeanin AHDAC5.95 ± 0.005.38 ± 0.135.49 ± 0.02
666–15CREB5.88 ± 0.125.79 ± 0.035.46 ± 0.03
AbexinostatHDAC5.48 ± 0.005.26 ± 0.33< 5.00
MI2Menin-MLL5.48 ± 0.005.48 ± 0.00< 5.00
GivinostatHDAC5.35 ± 0.455.35 ± 0.18< 5.00
MMV019721P. falciparum ACS5.31 ± 0.035.25 ± 0.45< 5.00
CerdulatinibSYK/JAK5.33 ± 0.205.26 ± 0.31< 5.00
PracinostatHDAC5.32 ± 0.135.72 ± 0.20< 5.00
CCT241736FLT3/Aurora Kinase5.24 ± 0.335.24 ± 0.34< 5.00
CyproheptadineSETD5.24 ± 0.345.46 ± 0.03< 5.00

  1. HDAC: histone deacetylase. CREB: cAMP response element-binding protein. FLT3: fms-like tyrosine kinase 3. P. falciparum ACS: P. falciparum acetyl CoA synthetase. SYK: spleen tyrosine kinase. JAK: Janus kinase. SETD: SET domain containing histone lysine methyltransferase. Mean and standard deviation are from two or more independent experiments.

Table 2—source data 1

Source data for Table 2.

https://cdn.elifesciences.org/articles/98221/elife-98221-table2-data1-v1.xlsx

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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Discussion

Herein we demonstrate several significant advances that progress radical cure antimalarial drug discovery and development, including the first report of screening a medium-sized (>10,000) compound library against mature hypnozoites as well as detection of novel hits with mechanisms unrelated to that of 8-aminoquinolines. Identification of these hits was made possible following the establishment of a complex logistical operation in which the sporozoites used for screening were produced by feeding P. vivax-infected blood from malaria patient isolates to mosquito colonies at malaria research institutes in two countries in Southeast Asia. Our international collaboration overcame several logistical hurdles to obtain positive Z-factors for most screening plates. Hits were also confirmed via dose–response, indicating that expanded screening directed against P. vivax liver stages is likely to produce more hypnozoite-specific hits (Table 1).

The only class of FDA-approved compounds for radical cure, the 8-aminoquinolines, was not discovered from in vitro drug screening. Instead, they were discovered using animal models, including the P. cynomolgi-infected rhesus macaque system (Rangel and Llinás, 2021). The 8-aminoquinolines function through generation of reactive oxygen species affecting both the host and parasite and lack a distinct parasite target (Dong et al., 2022; Watson et al., 2022; Camarda et al., 2019; Davidson et al., 1981). As such, this work represents one of the first applications of a radical cure development pipeline to begin with in vitro screening against P. vivax hypnozoites and end with attempted confirmation using P. cynomolgi radical cure models. While our screen generated positive results against P. vivax, we found mixed results against P. cynomolgi hypnozoites in vitro (Figure 1B, Supplementary file 2). While further studies will be needed to confirm that targets of our hits are parasite- or host-directed, our data show there is sufficient diversity in gene expression, structural biology, or mechanisms of hepatic quiescence between P. cynomolgi and P. vivax hypnozoites that some newly identified hits may be species-specific. While this result could also be attributed to differential metabolism in human and monkey hepatocytes (Liang et al., 2020), the rhesus macaque radical cure model is currently considered an important prerequisite for continued drug development, including efficacy testing in controlled human infections. The role of this model in the radical cure drug development cascade may need to be reevaluated as some compounds identified as promising for the radical cure of P. vivax may be abandoned too quickly due to the lack of activity against P. cynomolgi. This result highlights the need for further development and validation of P. vivax-specific animal models (Flannery et al., 2022). Furthermore, this report adds to the broader discussion surrounding the successes and challenges of drug repurposing (Krishnamurthy et al., 2022). While direct repositioning of a known drug as a safe treatment for a new indication is the ideal outcome, it can serve as advanced starting points for further optimization and still has the potential for reducing the time and cost involved in developing an efficacious therapy.

In addition to the identification of promising new hits and direction, our data suggests that epigenetic control of pathogenic dormancy via DNA methylation is a pathway that could be potentially targeted by future antimalarials. This pathway has already been described for several disease agents capable of dormancy, including cancer cells (Ferrer et al., 2020) and tuberculosis (Shell et al., 2013). DNA methylation has also been validated as controlling critical processes in plants, which share evolutionary traits with Plasmodium (Merrick, 2021). DNA methylation in the genus Plasmodium was first described in P. falciparum blood stages (Ponts et al., 2013) and has been associated with gene expression, transcriptional elongation, and parasite growth (Lucky et al., 2023; Hammam et al., 2021; Lenz et al., 2024). Previous experiments have shown that hydralazine can directly inhibit DNA methylation in nuclear extracts of blood stage parasites but also inhibit a recombinant functional fragment of the P. falciparum DNMT (Ponts et al., 2013). We pursued several biomolecular approaches to confirm that cadralazine may also interact with P. vivax DNMT in liver stage parasites. Due to technical limitations, we used a two-drug combination study in which the known DNMT inhibitor 5-azacytidine potentiated cadralazine against P. vivax hypnozoites (Figure 2). While we continue to develop new protocols and confirm the direct interaction of cadralazine with P. vivax, we successfully confirmed 5mC marks in P. vivax and P. cynomolgi liver stage parasites using both immunofluorescence and whole genome bisulfite sequencing assays (Figures 3 and 4).

The current model of hypnozoite quiescence suggests RNA-binding proteins (RBPs) drive hypnozoite formation by preventing translation of target mRNAs associated with schizogony (Toenhake et al., 2023). In this model, histone acetylation results in euchromatin at the loci of RBPs, resulting in their expression and ongoing quiescence. Hypothetically, HDAC inhibitors would favor quiescence, while a treatment that decreases histone acetylation would favor schizogony. This model somewhat contrasts with our present findings that HDAC inhibitors and the ACS inhibitor MMV019721 successfully kill hypnozoites in vitro (Table 2). It is, however, likely that the identified RBPs are part of broader gene networks which, when perturbed by sudden modulation of epigenetic features such as DNA methylation and histone acetylation, result in a lethal level of dysregulation. While we still need to develop P. vivax transgenic lines to successfully study hypnozoite biology and further validate potential drug targets (Voorberg-van der Wel et al., 2020; Wel et al., 2021), the chemical probes that we described in this report could be used in combination with single-cell technology to more precisely perturb hypnozoites and refine our understanding of epigenetic pathways regulating hypnozoite formation and survival.

Materials and methods

ReFRAME library description and plating

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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).

Ethical approval for human subjects and animal use

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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).

ReFRAME primary screen against P. vivax liver stages

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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.

Normalization, hit selection, and dose–response confirmation in P. vivax liver stage assays

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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).

Hit confirmation in P. cynomolgi liver stage assays at UGA

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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.

Hit confirmation in P. cynomolgi and P. vivax liver stage assays at NITD

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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).

Confirmed hit counterscreens: P. falciparum asexual blood stage at Calibr

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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.

Confirmed hit counterscreens: P. falciparum asexual blood stage at UGA

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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.

Confirmed hit counterscreens: P. cynomolgi asexual blood stage at UGA

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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.

Confirmed hit counterscreens: P. berghei liver stage at Calibr

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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-LUCCON 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.

Confirmed hit counterscreens: mammalian cell cytotoxicity at Calibr

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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).

Combination drug studies in P. vivax liver stages

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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).

Immunofluorescent staining of methyl-cytosine modifications in P. vivax liver stages

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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).

Immunofluorescent staining of methyl-cytosine modifications in P. cynomolgi liver stages

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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).

Collection of P. vivax and P. cynomolgi sporozoites for methyl-cytosine characterization

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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.

Quantification of 5mC, 5hmC, and 2′-deoxyguanosine (dG) in genomic DNA by LC–MS/MS/MS

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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°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.

Extraction of nuclear protein

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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 assay

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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.

Bisulfite conversion and library preparation

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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.

DNA methylation analysis

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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.

Assessment of effect of 1-ABT on hepatic cytochrome P450 3A4 activity

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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.

Assessment of effect of 1-ABT on hepatic metabolism using mass spectrometry

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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.

Additional ReFRAME hit confirmation using an improved P. vivax liver stage assay

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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.

Epigenetic inhibitor library screen against P. vivax liver stages

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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.

Appendix 1

Immunofluorescent staining of 5mC and 5hmC in P. vivax blood stage parasites

An immunofluorescent staining approach has been used to detect both 5mC and 5hmC in P. falciparum blood stage parasites (Lucky et al., 2023), 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 Mondulkiri, Eastern Cambodia. The presence of P. vivax was determined using an RDT (CareStartTM Malaria Pf/pan RDTs, Accessbio) or microscopy, and monoinfections were confirmed by RT-PCR using species-specific primers (Canier et al., 2013). Venous blood used was collected in lithium heparin tubes and immediately processed on-site in a mobile laboratory. Leukocytes were depleted using NWF filters (Li et al., 2017). The leukocyte-depleted parasitized red blood cells were cryopreserved using glycerolyte 57 solution (Baxter) and immediately stored in liquid nitrogen (Russell et al., 2011). Blood isolates were thawed by addition of 12%, then 1.6%, and then 0.9% (wt/vol) 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 (Rangel et al., 2018) followed by a wash with RPMI and two washes with 1× PBS. Parasites were then fixed with 3% (vol/vol) paraformaldehyde and 0.01% (vol/vol) glutaraldehyde in 1× PBS for 1 hr at 4°C. After fixation, blood stage parasites 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. While we did detect 5mC and 5hmC methylation in residual human white blood cells, we could not confirm positive 5mC or 5hmC staining in P. vivax blood stage parasites from these isolates (Appendix 1—figure 1). 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 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.

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Cell line (Homo sapiens, female)HEK293TATCCATCC cat:CRL-3216; RRID:CVCL_0063Transformed fetal cells
Cell line (Homo sapiens, male)HepG2ATCCATCC cat:HB-8065; RRID:CVCL_0027Hepatoblastoma
Biological sample (Homo sapiens, male)Primary human hepatocytesBioIVTLot:UBVCryopreserved cryoplateable
Biological sample (Homo sapiens, male)Primary human hepatocytesBioIVTLot:BGWCryopreserved cryoplateable
Biological sample (Homo sapiens, male)Primary human hepatocytes, femaleBioIVTLot:QWKCryopreserved cryoplateable
Biological sample (Macaca fascicularis, male)Primary simian hepatocytesBioIVTLot:CWPCryopreserved cryoplateable
Biological sample (Macaca fascicularis, male)Primary simian hepatocytesBioIVTLot:NPICryopreserved cryoplateable
Biological sample (Macaca fascicularis, male)Primary simian hepatocytesBioIVTLot:NDOCryopreserved cryoplateable
Biological sample (Macaca mulatta, male)Primary simian hepatocytesBioIVTLot:XXJCryopreserved cryoplateable
Biological sample (Macaca mulatta, male)Primary simian hepatocytesBioIVTLot:NNFCryopreserved cryoplateable
Biological sample (An. dirus)MosquitoesShoklo Malaria Research UnitColony maintained on site
Biological sample (An. dirus)MosquitoesInstitute Pasteur of CambodiaColony maintained on site
Biological sample (An. dirus)MosquitoesArmed Forces Research Institute of Medical SciencesColony maintained on site
Biological sample (An. dirus)MosquitoesUniversity of GeorgiaColony maintained on site
Biological sample (Anopheles stephensi)MosquitoesUniversity of GeorgiaColony maintained and infected at UGA, shipped to Calibr for P. berghei assays
Biological sample (Plasmodium vivax)Patient isolateShoklo Malaria Research UnitPID:402389Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateShoklo Malaria Research UnitPID:423955Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateShoklo Malaria Research UnitPID:425583Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateShoklo Malaria Research UnitPID:432054Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateShoklo Malaria Research UnitPID:2020-013Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateShoklo Malaria Research UnitPID:2020-014Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateInstitute Pasteur of CambodiaPID:Pv593Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateInstitute Pasteur of CambodiaPID:Pv595Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateInstitute Pasteur of CambodiaPID:Pv602Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateInstitute Pasteur of CambodiaPID:Pv603Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateInstitute Pasteur of CambodiaPID:Pv606Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateInstitute Pasteur of CambodiaPID:Pv608Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateInstitute Pasteur of CambodiaPID:Pv609Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateInstitute Pasteur of CambodiaPID:Pv611Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateInstitute Pasteur of CambodiaPID:Pv623Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateInstitute Pasteur of CambodiaPID:Pv624Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateInstitute Pasteur of CambodiaPID:Pv635Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateInstitute Pasteur of CambodiaPID:Pv640Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateInstitute Pasteur of CambodiaPID:Pv644Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateInstitute Pasteur of CambodiaPID:Pv708Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateInstitute Pasteur of CambodiaPID:Pv836Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateInstitute Pasteur of CambodiaPID:Pv838Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateInstitute Pasteur of CambodiaPID:Pv846Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateInstitute Pasteur of CambodiaPID:Pv847Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateInstitute Pasteur of CambodiaPID:Pv849Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateInstitute Pasteur of CambodiaPID:Pv893Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateInstitute Pasteur of CambodiaPID:Pv922Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateInstitute Pasteur of CambodiaPID:Pv923Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateInstitute Pasteur of CambodiaPID:Pv950Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateInstitute Pasteur of CambodiaPID:Pv951Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateInstitute Pasteur of CambodiaPID:Pv952Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateInstitute Pasteur of CambodiaPID:Pv959Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateInstitute Pasteur of CambodiaPID:Pv1014Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateInstitute Pasteur of CambodiaPID:Pv1020Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateInstitute Pasteur of CambodiaPID:Pv1024Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateInstitute Pasteur of CambodiaPID:IV21-075Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateInstitute Pasteur of CambodiaPID:PQRC21-113Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateInstitute Pasteur of CambodiaPID:PQRC21-135Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium vivax)Patient isolateMahidol Vivax Research UnitPID:VTTY201Fresh isolate fed to An. dirus mosquitoes
Biological sample (Plasmodium cynomolgi)M/B strainPMID:31536608Emory National Primate Research Center
Biological sample (Plasmodium cynomolgi)Rossan strainPMID:18788885Emory National Primate Research Center
Biological sample (Plasmodium cynomolgi)B strainPMID:32660993Armed Forces Research Institute of Medical Sciences
Biological sample (Plasmodium cynomolgi)P. berghei ANKA strain GFP Lucama1-eef1a (line 1052cl1)PMID:36100902University of Georgia
Biological sample (Plasmodium falciparum)Dd2-HLHBEI ResourcesCat#:MRA-156
Biological sample (Plasmodium cynomolgi)DCThis paperUniversity of Georgia
Biological sample (Plasmodium falciparum)W2PMID:7729473University of Georgia
Strain, strain background (Macaca fuscata, male)Monkey, used for experimental animal infectionEmory National Primate Research CenterNot genetically modified
Strain, strain background (Macaca fuscata, male)Monkey, used for experimental animal infectionEmory National Primate Research CenterNot genetically modified
Antibodyanti P. vivax Upregulated in Infectious Sporozoites 4 (rPvUIS4) (recombinant mouse monoclonal)PMID:30333026IFA (1:10,000)
Antibodyanti-PvMIF (rabbit polyclonal)PMID:25800544IFA (1:1000)
Antibodyanti-PcHSP70 (rabbit polyclonal)This papern/aIFA (200 ng/ml)
Antibodyanti-PcUIS4 (mouse monoclonal)This papern/aIFA (10 ng/ml)
Antibodyanti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Goat monoclonal)Thermo Fisher ScientificCat#: A-11001; RRID:AB_2534069IFA (1:1000)
Antibodyanti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Goat monoclonal)Thermo Fisher ScientificCat#:A11013; RRID:AB_2534080IFA (1:1000)
Antibodyanti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 (Goat monoclonal)Thermo Fisher ScientificCat#:A11036; RRID:AB_10563566IFA (1:1000)
Antibody5-Methylcytosine Recombinant Antibody (rabbit monoclonal)Thermo Fisher ScientificCat#:MA5-24694: RRID:AB_2665309; Clone:RM23110 μg/ml
Antibody5-Hydroxymethylcytosine Recombinant Antibody (rabbit monoclonal)Thermo Fisher ScientificCat#:MA5-24695; RRID:AB_2665308; Clone:RM23610 μg/ml
Antibodyanti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Texas Red (goat monoclonal)Thermo Fisher ScientificCat#:T-2767; RRID:AB_255677610 μg/ml
Antibodyanti-Plasmodium GAPDH (mouse monoclonal)European Malaria Reagent RepositoryCat#:13.3100 ng/ml
Software, algorithmGenedata Screener, Version 15.0.1-StandardGenedata
Chemical compound, drugBudralazineChemcruzCat3:sc-504334Batch D3019
Chemical compound, drugCadralazineChemcruzCat#:sc-500641Batch B24217
Chemical compound, drugHydralazineSelleckchemCat#:S2562Batch S256202
Chemical compound, drugDihydralazineCalibr at ScrippsCode:CBR-001-571-820-4
Chemical compound, drugPlasmocidCalibr at ScrippsCode:CBR-001-572-110-5
Chemical compound, drugMS-0735Calibr at ScrippsCode:CBR-001-572-134-3
Chemical compound, drugHydralazineCalibr at ScrippsCode:CBR-001-572-134-3
Chemical compound, drugColforsin daropateCalibr at ScrippsCode:CBR-001-586-408-1
Chemical compound, drugPAN-811Calibr at ScrippsCode:CBR-001-586-749-9
Chemical compound, drugTodralazineCalibr at ScrippsCode:CBR-001-586-916-6
Chemical compound, drugRGH-5526Calibr at ScrippsCode:CBR-001-587-032-3
Chemical compound, drugBudralazineCalibr at ScrippsCode:CBR-001-587-246-5
Chemical compound, drugDramedilolCalibr at ScrippsCode:CBR-001-593-286-2
Chemical compound, drugEndralazineCalibr at ScrippsCode:CBR-001-597-262-0
Chemical compound, drugCadralazineCalibr at ScrippsCode:CBR-001-624-776-0
Chemical compound, drugPildralazineCalibr at ScrippsCode:CBR-001-635-378-9
Chemical compound, drugMopidralazineCalibr at ScrippsCode:CBR-001-635-852-4
Chemical compound, drugRhodamine 123Calibr at ScrippsCode:CBR-050-127-020-8
Chemical compound, drugNarasinCalibr at ScrippsCode:CBR-050-127-705-0
Chemical compound, drugPoziotinibCalibr at ScrippsCode:CBR-001-574-260-6
Chemical compound, drugPanobinostatTargetmolCat#:T2383
Chemical compound, drugAbexinostatTargetmolCat#:T0431
Chemical compound, drugPracinostatTargetmolCat#:T1890
Chemical compound, drugCyproheptadineTargetmolCat#:T0174
Chemical compound, drugCerdulatinibTargetmolCat#:T2487
Chemical compound, drugMI2TargetmolCat#:T2649
Chemical compound, drugRaddeanin ATargetmolCat#:T3878
Chemical compound, drugCCT241736TargetmolCat#:T4428
Chemical compound, drug666-15TargetmolCat#:T5318
Chemical compound, drugGivinostatTargetmolCat#:T6279
Chemical compound, drugAR42TargetmolCat#:T6392
Chemical compound, drugMMV019721Medicines for Malaria VentureCode:MMV019721Batch:MMV019721-08, MMV019721-10
Chemical compound, drugMMV084978Medicines for Malaria VentureCode:MMV084978Batch:MMV084978-04, MMV084978-05
Chemical compound, drug5-AzacytidineCyamen ChemCat#:11164
Chemical compound, drug1-AminobenzotriazoleCyamen ChemCat#:15252
Commercial assay or kitCell-Titer GloPromegaCat#:G7573
Commercial assay or kitEpiQuik DNA Methyltransferase (DNMT) Activity/Inhibition Assay KitEpiGentekCat#:P-3010
Commercial assay or kitEpitect fast bisulfite conversion kitQIAGENCat#:59824
Commercial assay or kitCYP3A4 luciferin-IPA kitPromegaCat#:V9001Used Lytic protocol
Appendix 1—figure 1
Download asset Open asset
Cytosine modification in P. vivax blood stages.

(A) P. vivax blood stages from patient isolates appeared negative when stained with 5mC. A white blood cell positive for 5mC serves as a stain control. (B) P. vivax blood stages from patient isolates appeared negative when stained with 5hmC. A white blood cell positive for 5hmC serves as a stain control. Bars represent 10 µm.

Data availability

All bisulfite sequencing data generated in this study can be found in the Sequence Read Archive (SRA) at the NCBI National Library of Medicine (https://www.ncbi.nlm.nih.gov/sra) under the BioProject code PRJNA925570.

The following data sets were generated
    1. Gupta M
    2. Lenz T
    3. Prudhomme J
    4. Le Roch KG
    (2024) NCBI BioProject
    ID PRJNA925570. A Drug Repurposing Approach Reveals Targetable Epigenetic Pathways in Plasmodium vivax Hypnozoites.
    https://www.ncbi.nlm.nih.gov/bioproject/PRJNA925570/

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    Journal of Immunology 140:2197–2200.
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    New tissue schizontocidal antimalarial drugs
    Bulletin of the World Health Organization 59:463–479.
    1. Lambros C
    2. Vanderberg JP
    (1979)
    Synchronization of Plasmodium falciparum erythrocytic stages in culture
    The Journal of Parasitology 65:418–420.
  55. Book
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Article and author information

Author details

  1. Steven P Maher

    Center for Tropical and Emerging Global Disease, University of Georgia, Athens, United States
    Contribution
    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing
    For correspondence
    STEVEN.MAHER@uga.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9560-5656

  2. Malina A Bakowski

    Calibr, a division of The Scripps Research Institute, La Jolla, United States
    Contribution
    Conceptualization, Resources, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3337-6528

  3. Amélie Vantaux

    Malaria Molecular Epidemiology Unit, Institute Pasteur of Cambodia, Phnom Penh, Cambodia
    Contribution
    Conceptualization, Resources, Supervision, Funding acquisition, Investigation, Methodology, Project administration, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7945-961X

  4. Erika L Flannery

    Novartis Institute for Tropical Diseases, Novartis Institutes for Biomedical Research, Emoryville, United States
    Contribution
    Data curation, Supervision, Validation, Investigation, Writing – review and editing
    Competing interests
    AH-C, VC, ELF, and SAM are employees of the Novartis Institute for Tropical Disease
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0665-7954

  5. Chiara Andolina

    Shoklo Malaria Research Unit, Mahidol-Oxford Tropical Medicine Research Unit, Mae Sot, Thailand
    Contribution
    Resources
    Competing interests
    No competing interests declared

  6. Mohit Gupta

    Department of Molecular, Cell, and Systems Biology, University of California, Riverside, Riverside, United States
    Contribution
    Supervision, Investigation
    Competing interests
    No competing interests declared

  7. Yevgeniya Antonova-Koch

    Calibr, a division of The Scripps Research Institute, La Jolla, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared

  8. Magdalena Argomaniz

    Center for Vaccines and Immunology, College of Veterinary Medicine, University of Georgia, Athens, United States
    Contribution
    Resources
    Competing interests
    No competing interests declared

  9. Monica Cabrera-Mora

    International Center for Malaria Research, Education and Development, Emory Vaccine Center, Emory National Primate Research Center, Emory University, Atlanta, United States
    Contribution
    Resources
    Competing interests
    No competing interests declared

  10. Brice Campo

    Medicines for Malaria Venture (MMV), Geneva, Switzerland
    Contribution
    Supervision
    Competing interests
    BC is an employee of MMV

  11. Alexander T Chao

    Novartis Institute for Tropical Diseases, Novartis Institutes for Biomedical Research, Emoryville, United States
    Contribution
    Data curation, Investigation
    Competing interests
    No competing interests declared

  12. Arnab K Chatterjee

    Calibr, a division of The Scripps Research Institute, La Jolla, United States
    Contribution
    Supervision
    Competing interests
    No competing interests declared

  13. Wayne T Cheng

    Center for Vaccines and Immunology, College of Veterinary Medicine, University of Georgia, Athens, United States
    Contribution
    Resources
    Competing interests
    No competing interests declared

  14. Vorada Chuenchob

    Novartis Institute for Tropical Diseases, Novartis Institutes for Biomedical Research, Emoryville, United States
    Contribution
    Investigation
    Competing interests
    AH-C, VC, ELF, and SAM are employees of the Novartis Institute for Tropical Disease,

  15. Caitlin A Cooper

    Center for Tropical and Emerging Global Disease, University of Georgia, Athens, United States
    Contribution
    Resources, Investigation, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared

  16. Karissa Cottier

    BioIVT Inc, New York, United States
    Contribution
    Investigation, Writing – review and editing
    Competing interests
    TM and KC are employees of BioIVT

  17. Mary R Galinski

    1. International Center for Malaria Research, Education and Development, Emory Vaccine Center, Emory National Primate Research Center, Emory University, Atlanta, United States
    2. Division of Infectious Diseases, Department of Medicine, Emory University, Atlanta, United States
    Contribution
    Supervision, Investigation
    Competing interests
    No competing interests declared

  18. Anke Harupa-Chung

    Novartis Institute for Tropical Diseases, Novartis Institutes for Biomedical Research, Emoryville, United States
    Contribution
    Data curation, Investigation
    Competing interests
    AH-C, VC, ELF, and SAM are employees of the Novartis Institute for Tropical Disease

  19. Hana Ji

    Center for Vaccines and Immunology, College of Veterinary Medicine, University of Georgia, Athens, United States
    Contribution
    Resources
    Competing interests
    No competing interests declared

  20. Sean B Joseph

    Calibr, a division of The Scripps Research Institute, La Jolla, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared

  21. Todd Lenz

    Department of Molecular, Cell, and Systems Biology, University of California, Riverside, Riverside, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared

  22. Stefano Lonardi

    Department of Computer Science and Engineering, University of California, Riverside, Riverside, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared

  23. Jessica Matheson

    Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand
    Contribution
    Investigation, Visualization
    Competing interests
    No competing interests declared

  24. Sebastian A Mikolajczak

    Novartis Institute for Tropical Diseases, Novartis Institutes for Biomedical Research, Emoryville, United States
    Contribution
    Supervision
    Competing interests
    AH-C, VC, ELF, and SAM are employees of the Novartis Institute for Tropical Disease

  25. Timothy Moeller

    BioIVT Inc, New York, United States
    Contribution
    Investigation
    Competing interests
    TM and KC are employees of BioIVT

  26. Agnes Orban

    Malaria Molecular Epidemiology Unit, Institute Pasteur of Cambodia, Phnom Penh, Cambodia
    Contribution
    Investigation
    Competing interests
    No competing interests declared

  27. Vivian Padín-Irizarry

    1. Center for Tropical and Emerging Global Disease, University of Georgia, Athens, United States
    2. School of Sciences, Clayton State University, Morrow, United States
    Contribution
    Investigation, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared

  28. Kastin Pan

    Calibr, a division of The Scripps Research Institute, La Jolla, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0009-0003-5838-1694

  29. Julie Péneau

    Malaria Molecular Epidemiology Unit, Institute Pasteur of Cambodia, Phnom Penh, Cambodia
    Contribution
    Investigation
    Competing interests
    No competing interests declared

  30. Jacques Prudhomme

    Department of Molecular, Cell, and Systems Biology, University of California, Riverside, Riverside, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6161-5194

  31. Camille Roesch

    Malaria Molecular Epidemiology Unit, Institute Pasteur of Cambodia, Phnom Penh, Cambodia
    Contribution
    Resources, Investigation, Visualization, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared

  32. Anthony Ruberto

    Center for Tropical and Emerging Global Disease, University of Georgia, Athens, United States
    Contribution
    Formal analysis
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3215-9484

  33. Saniya S Sabnis

    Center for Vaccines and Immunology, College of Veterinary Medicine, University of Georgia, Athens, United States
    Contribution
    Resources
    Competing interests
    No competing interests declared

  34. Celia L Saney

    Center for Vaccines and Immunology, College of Veterinary Medicine, University of Georgia, Athens, United States
    Contribution
    Resources
    Competing interests
    No competing interests declared

  35. Jetsumon Sattabongkot

    Mahidol Vivax Research Unit, Mahidol University, Bangkok, Thailand
    Contribution
    Resources
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3938-4588

  36. Saleh Sereshki

    Department of Computer Science and Engineering, University of California, Riverside, Riverside, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared

  37. Sangrawee Suriyakan

    Shoklo Malaria Research Unit, Mahidol-Oxford Tropical Medicine Research Unit, Mae Sot, Thailand
    Contribution
    Investigation
    Competing interests
    No competing interests declared

  38. Ratawan Ubalee

    Department of Entomology, Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok, Thailand
    Contribution
    Resources
    Competing interests
    No competing interests declared

  39. Yinsheng Wang

    1. Department of Chemistry, University of California, Riverside, Riverside, United States
    2. Environmental Toxicology Graduate Program, University of California, Riverside, Riverside, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared

  40. Praphan Wasisakun

    Shoklo Malaria Research Unit, Mahidol-Oxford Tropical Medicine Research Unit, Mae Sot, Thailand
    Contribution
    Resources
    Competing interests
    No competing interests declared

  41. Jiekai Yin

    Environmental Toxicology Graduate Program, University of California, Riverside, Riverside, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared

  42. Jean Popovici

    Malaria Molecular Epidemiology Unit, Institute Pasteur of Cambodia, Phnom Penh, Cambodia
    Contribution
    Resources, Supervision
    Competing interests
    No competing interests declared

  43. Case W McNamara

    Calibr, a division of The Scripps Research Institute, La Jolla, United States
    Contribution
    Conceptualization
    Competing interests
    No competing interests declared

  44. Chester Joyner

    1. Center for Vaccines and Immunology, College of Veterinary Medicine, University of Georgia, Athens, United States
    2. International Center for Malaria Research, Education and Development, Emory Vaccine Center, Emory National Primate Research Center, Emory University, Atlanta, United States
    Contribution
    Resources, Supervision, Funding acquisition, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1367-2829

  45. François H Nosten

    1. Shoklo Malaria Research Unit, Mahidol-Oxford Tropical Medicine Research Unit, Mae Sot, Thailand
    2. Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
    Contribution
    Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7951-0745

  46. Benoît Witkowski

    Malaria Molecular Epidemiology Unit, Institute Pasteur of Cambodia, Phnom Penh, Cambodia
    Contribution
    Resources, Supervision, Funding acquisition, Project administration
    Competing interests
    No competing interests declared

  47. Karine G Le Roch

    Department of Molecular, Cell, and Systems Biology, University of California, Riverside, Riverside, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing
    Competing interests
    No competing interests declared

  48. Dennis E Kyle

    Center for Tropical and Emerging Global Disease, University of Georgia, Athens, United States
    Contribution
    Supervision, Funding acquisition, Project administration, Writing – review and editing
    For correspondence
    Dennis.Kyle@uga.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0238-965X

Funding

Bill and Melinda Gates Foundation (#OPP1107194)

  • Malina A Bakowski
  • Case W McNamara

Bill and Melinda Gates Foundation (INV-031788)

  • Chester Joyner

Bill and Melinda Gates Foundation (#OPP1023601)

  • Dennis E Kyle

Medicines for Malaria Venture (RD/17/0042)

  • Amélie Vantaux
  • Benoît Witkowski

Medicines for Malaria Venture (RD/15/0022)

  • Steven P Maher
  • Amélie Vantaux
  • Benoît Witkowski
  • Dennis E Kyle

National Institutes of Health (#HHSN272201200031C)

  • Mary R Galinski

National Institutes of Health (1R01 AI136511)

  • Karine G Le Roch

University of California, Riverside (#NIFA-Hatch-225935)

  • Karine G Le Roch

The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication. For the purpose of Open Access, the authors have applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission

Acknowledgements

We thank the malaria patients of Thailand and Cambodia for participation in this study. We thank the Sporocore at UGA for generating P. berghei-infected mosquitoes. We are grateful to Calibr’s Compound Management and High Throughput Screening Groups for their assistance with this project. HCI data from drug studies was produced by the Biomedical Microscopy Core at UGA, supported by the Georgia Research Alliance. SMRU is part of the Mahidol Oxford Research Unit, supported by the Wellcome Trust of Great Britain (#220211). Material has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation and/or publication. The opinions or assertions contained herein are the private views of the author, and are not to be construed as official or as reflecting true views of the Department of the Army or the Department of Defense. This publication includes data generated at the University of California, San Diego IGM Genomics Center utilizing an Illumina NovaSeq 6000 that was purchased with funding from a National Institutes of Health SIG grant (#S10 OD026929). Funding support was provided by the Bill & Melinda Gates Foundation (#OPP1107194 to Calibr, INV-031788 to CJJ, and #OPP1023601 to DEK), Medicines for Malaria Venture (RD/17/0042 and RD/15/0022 to BW and AV and RD/15/0022 to SPM and DEK), the National Institutes of Allergy and Infectious Diseases of the National Institutes of Health (#HHSN272201200031C to MRG and #1R01 AI136511 to KGLR), and the University of California, Riverside (#NIFA-Hatch-225935 to KGLR).

Ethics

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 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.

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© 2024, Maher et al.

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  1. Steven P Maher
  2. Malina A Bakowski
  3. Amélie Vantaux
  4. Erika L Flannery
  5. Chiara Andolina
  6. Mohit Gupta
  7. Yevgeniya Antonova-Koch
  8. Magdalena Argomaniz
  9. Monica Cabrera-Mora
  10. Brice Campo
  11. Alexander T Chao
  12. Arnab K Chatterjee
  13. Wayne T Cheng
  14. Vorada Chuenchob
  15. Caitlin A Cooper
  16. Karissa Cottier
  17. Mary R Galinski
  18. Anke Harupa-Chung
  19. Hana Ji
  20. Sean B Joseph
  21. Todd Lenz
  22. Stefano Lonardi
  23. Jessica Matheson
  24. Sebastian A Mikolajczak
  25. Timothy Moeller
  26. Agnes Orban
  27. Vivian Padín-Irizarry
  28. Kastin Pan
  29. Julie Péneau
  30. Jacques Prudhomme
  31. Camille Roesch
  32. Anthony Ruberto
  33. Saniya S Sabnis
  34. Celia L Saney
  35. Jetsumon Sattabongkot
  36. Saleh Sereshki
  37. Sangrawee Suriyakan
  38. Ratawan Ubalee
  39. Yinsheng Wang
  40. Praphan Wasisakun
  41. Jiekai Yin
  42. Jean Popovici
  43. Case W McNamara
  44. Chester Joyner
  45. François H Nosten
  46. Benoît Witkowski
  47. Karine G Le Roch
  48. Dennis E Kyle
(2025)
A drug repurposing approach reveals targetable epigenetic pathways in Plasmodium vivax hypnozoites
eLife 13:RP98221.
https://doi.org/10.7554/eLife.98221.2

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