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

Reviewer #1 (Public Review):

Summary:

Plasmodium vivax can persist in the liver of infected individuals in the form of dormant hypnozoites, which cause malaria relapses and are resistant to most current antimalarial drugs. This highlights the need to develop new drugs active against hypnozoites that could be used for radical cure. Here, the authors capitalize on an in vitro culture system based on primary human hepatocytes infected with P. vivax sporozoites to screen libraries of repurposed molecules and compounds acting on epigenetic pathways. They identified a number of hits, including hydrazinophthalazine analogs. They propose that some of these compounds may act on epigenetic pathways potentially involved in parasite quiescence. To provide some support to this hypothesis, they document DNA methylation of parasite DNA based on 5-methylcytosine immunostaining, mass spectrometry, and bisulfite sequencing.

Strengths:
-The drug screen itself represents a huge amount of work and, given the complexity of the experimental model, is a tour de force.
-The screening was performed in two different laboratories, with a third laboratory being involved in the confirmation of some of the hits, providing strong support that the results were reproducible.
-The screening of repurposing libraries is highly relevant to accelerate the development of new radical cure strategies.

Weaknesses:

-The manuscript is composed of two main parts, the drug screening itself and the description of DNA methylation in Plasmodium pre-erythrocytic stages. Unfortunately, these two parts are loosely connected. First, there is no evidence that the identified hits kill hypnozoites via epigenetic mechanisms. The hit compounds almost all act on schizonts in addition to hypnozoites, therefore it is unlikely that they target quiescence-specific pathways. At least one compound, colforsin, seems to selectively act on hypnozoites, but this observation still requires confirmation. Second, while the description of DNA methylation is per se interesting, its role in quiescence is not directly addressed here. Again, this is clearly not a specific feature of hypnozoites as it is also observed in P. vivax and P. cynomolgi hepatic schizonts and in P. falciparum blood stages. Therefore, the link between DNA methylation and hypnozoite formation is unclear. In addition, DNA methylation in sporozoites may not reflect epigenetic regulation occurring in the subsequent liver stages.

-The mode of action of the hit compounds remains unknown. In particular, it is not clear whether the drugs act on the parasite or on the host cell. Merely counting host cell nuclei to evaluate the toxicity of the compounds is probably acceptable for the screen but may not be sufficient to rule out an effect on the host cell. A more thorough characterization of the toxicity of the selected hit compounds is required.

-There is no convincing explanation for the differences observed between P. vivax and P. cynomolgi. The authors question the relevance of the simian model but the discrepancy could also be due to the P. vivax in vitro platform they used.

-Many experiments were performed only once, not only during the screen (where most compounds were apparently tested in a single well) but also in other experiments. The quality of the data would be increased with more replication.

-While the extended assay (12 days versus 8 days) represents an improvement of the screen, the relevance of adding inhibitors of core cytochrome activity is less clear, as under these conditions the culture system deviates from physiological conditions.

Reviewer #2 (Public Review):

Summary:

In this manuscript, inhibitors of the P. vivax liver stages are identified from the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) library as well as a 773-member collection of epigenetic inhibitors. This study led to the discovery that epigenetics pathway inhibitors are selectively active against P. vivax and P. cynomolgi hypnozoites. Several inhibitors of histone post-translational modifications were found among the hits and genomic DNA methylation mapping revealed the modification on most genes. Experiments were completed to show that the level of methylation upstream of the gene (promoter or first exon) may impact gene expression. With the limited number of small molecules that act against hypnozoites, this work is critically important for future drug leads. Additionally, the authors gleaned biological insights from their molecules to advance the current understanding of essential molecular processes during this elusive parasite stage.

Strengths:
-This is a tremendously impactful study that assesses molecules for the ability to inhibit Plasmodium hypnozoites. The comparison of various species is especially relevant for probing biological processes and advancing drug leads.

-The SI is wonderfully organized and includes relevant data/details. These results will inspire numerous studies beyond the current work.

Reviewer #3 (Public Review):

Although this work represents a massive screening effort to find new drugs targeting P. vivax hypnozoites, the authors should balance their statement that they identified targetable epigenetic pathways in hypnozoites.

• They should emphasize the potential role of the host cell in the presentation of the results and the discussion, as it is known that other pathogens modify the epigenome of the host cell (i.e. toxoplasma, HIV) to prevent cell division. Also, hydrazinophtalazines target multiple pathways (notably modulation of calcium flux) and have been shown to inhibit DNA-methyl transferase 1 which is lacking in Plasmodium.

• In a drug repurposing approach, the parasite target might also be different than the human target.

• The authors state that host-cell apoptotic pathways are downregulated in P. vivax infected cells (p. 5 line 162). Maybe the HDAC inhibitors and DNA-methyltransferase inhibitors are reactivating these pathways, leading to parasite death, rather than targeting parasites directly.

It would make the interpretation of the results easier if the authors used EC50 in µM rather than pEC50 in tables and main text. It is easy to calculate when it is a single-digit number but more complicated with multiple digits.

Authors mention hypnozoite-specific effects but in most cases, compounds are as potent on hypnozoite and schizonts. They should rather use "liver stage specific" to refer to increased activity against hypnozoites and schizonts compared to the host cell. The same comment applies to line 351 when referring to MMV019721. Following the same idea, it is a bit far-fetched to call MMV019721 "specific" when the highest concentration tested for cytotoxicity is less than twice the EC50 obtained against hypnozoites and schizonts.

Page 5 lines 187-189, the authors state "...hydrazinophtalazines were inactive when tested against P. berghei liver schizonts and P. falciparum asexual blood stages, suggesting that hypnozoite quiescence may be biologically distinct from developing schizonts". The data provided in Figure 1B show that these hydrazinophtalazines are as potent in P. vivax schizonts than in P. vivax hypnozoites, so the distinct activity seems to be Plasmodium species specific and/or host-cell specific (primary human hepatocytes rather than cell lines for P. berghei) rather than hypnozoite vs schizont specific.

Why choose to focus on cadralazine if abandoned due to side effects? Also, why test the pharmacokinetics in monkeys? As it was a marketed drug, were no data available in humans?

In the counterscreen mentioned on page 6, the authors should mention that the activity of poziotinib in P. berghei and P. cynomolgi is equivalent to cell toxicity, so likely not due to parasite specificity.

To improve the clarity and flow of the manuscript, could the authors make a recapitulative table/figure for all the data obtained for poziotinib and hydrazinophtalazines in the different assays (8-days vs 12-days) and laboratory settings rather than separate tables in main and supplementary figures. Maybe also reorder the results section notably moving the 12-day assay before the DNA methylation part.

The isobologram plot shows an additive effect rather than a synergistic effect between cadralazine and 5-azacytidine, please modify the paragraph title accordingly. Please put the same axis scale for both fractional EC50 in the isobologram graph (Figure 2A).

Concerning the immunofluorescence detection of 5mC and 5hmC, the authors should be careful with their conclusions. The Hoechst signal of the parasites is indistinguishable because of the high signal given by the hepatocyte nuclei. The signal obtained with the anti-5hmC in hepatocyte nuclei is higher than with the anti-5mC, thus if a low signal is obtained in hypnozoites and schizonts, it might be difficult to dissociate from the background. In blood stages (Figure S18), the best to obtain a good signal is to lyse the red blood cell using saponin, before fixation and HCl treatment.

To conclude that 5mC marks are the predominate DNA methylation mark in both P. falciparum and P. vivax, authors should also mention that they compare different stages of the life cycle, that might have different methylation levels.

Also, the authors conclude that "[...] 5mC is present at low level in P. vivax and P. cynomolgi sporozoites and could control liver stage development and hypnozoite quiescence". Based on the data shown here, nothing, except presence the of 5mC marks, supports that DNA methylation could be implicated in liver stage development or hypnozoite quiescence.

How many DNA-methyltransferase inhibitors were present in the epigenetic library? Out of those, none were identified as hits, maybe the hydrazinophtalazines effect is not linked to DNMT inhibition but another target pathway of these molecules like calcium transport?

The authors state (line 344): "These results corroborate our hypothesis that epigenetic pathways regulate hypnozoites". This conclusion should be changed to "[...] that epigenetic pathways are involved in P. vivax liver stage survival" because:
• The epigenetic inhibitors described here are as active on hypnozoite than liver schizonts.
• Again, we cannot rule out that the host cell plays a role in this effect and that the compound may not act directly on the parasite.

The same comment applies to the quote in lines 394 to 396. There is no proof in the results presented here that DNA methylation plays any role in the effect of hydrazinophtalazines in the anti-plasmodial activity obtained in the assay.