Malaria is a disease spread by mosquitoes. When infected insects bite the skin, they inject parasites called Plasmodium into the host. The symptoms of the disease then develop when Plasmodium infect host red blood cells. These parasites cannot make the raw materials to build their own proteins, so instead, they digest haemoglobin – the protein used by red blood cells to carry oxygen – and use its building blocks to produce proteins.

Blocking the digestion of haemoglobin can stop malaria infections in their tracks, but it is unclear how exactly Plasmodium parasites break down the protein. Researchers think that a group of four enzymes called aminopeptidases are responsible for the final stage in this digestion, releasing the amino acids that make up haemoglobin. However, the individual roles of each of these aminopeptidases are not yet known.

To start filling this gap, Edgar et al. set out to study one of these aminopeptidases, called PfA-M17. First, they genetically modified Plasmodium falciparum parasites so that the levels of this aminopeptidase were reduced during infection. Without the enzyme, the parasites were unable to grow. The next step was to confirm that this was because PfA-M17 breaks down haemoglobin, and not for another reason. To test this, Edgar et al. designed a new molecule that could stop PfA-M17 from releasing amino acids. This molecule, which they called ‘compound 3’, had the same effect as reducing the levels of PfA-M17. Further analysis showed that the amino acids that PfA- M17 releases match the amino acids found in haemoglobin.

Malaria causes hundreds of thousands of deaths per year. Although there are treatments available, the Plasmodium parasites are starting to develop resistance. Confirming the role of PfA-M17 provides a starting point for new studies by parasitologists, biologists, and drug developers. This could lead to the development of chemicals that block this enzyme, forming the basis for new treatments.

Malaria is an infectious disease caused by protozoan parasites belonging to the Plasmodium genus, of which Plasmodium falciparum is the deadliest to humans. In 2020, there were more than 600,000 deaths attributed to malaria infections, the majority of these occurring throughout sub-Saharan Africa and South-East Asia (WHO, 2021). The current front-line therapeutic artemisinin and its derivatives, as well as partner drugs used in combination therapies, are under threat as resistance to these drugs continues to arise. Artemisinin resistance, which was once localized to the Greater Mekong sub-region within Asia, has now been identified elsewhere, including most worryingly in malaria endemic regions of Africa, with de novo resistance identified in Rwanda and delayed parasite clearance times confirmed in Uganda (Uwimana et al., 2020; Uwimana et al., 2021). The threat of a growing spread of resistance highlights the need to identify new therapeutic targets and compounds with novel modes of action (Ataide et al., 2017).

The intra-erythrocytic cycle of P. falciparum is responsible for the clinical manifestations of disease and is the target of most antimalarial drugs. Parasite survival during the erythrocytic stage is dependent upon the digestion of host hemoglobin to provide amino acids essential for parasite growth, with the exception of isoleucine which is absent from human hemoglobin and, therefore, has to be taken up from the extracellular environment (Babbitt et al., 2012). Digestion of hemoglobin also creates space in the erythrocyte to accommodate the growing parasite, as well as providing a mechanism to regulate the osmotic pressure of the host cell (Krugliak et al., 2002; Lew et al., 2004; Liu et al., 2006; Rosenthal, 2002). Hemoglobin digestion begins during the early ring stage of growth, but formation of hemozoin crystals, a detoxified version of the digestive by-product heme, can only be visualized by microscopy in the more developed trophozoite stage in a specialized acidic compartment termed the digestive vacuole (DV) (Abu Bakar et al., 2010). The DV contains an array of proteases responsible for the release of peptides from hemoglobin, including plasmepsins, falcipains, falcilysin, and aminopeptidases Goldberg, 2005; it is the latter which are speculated to perform the final step of amino acid cleavage from the amino-terminal end of these peptides (Gavigan et al., 2001).

As aminopeptidases are implicated in the final step of hemoglobin digestion, they are promising novel therapeutic targets. The P. falciparum genome encodes two neutral metallo-aminopeptidases: the M1 alanyl aminopeptidase (PfA-M1; PfM1AAP) and M17 leucyl aminopeptidase (PfA-M17; PfM17LAP). The broad-spectrum aminopeptidase inhibitor bestatin has been shown to kill P. falciparum, with parasites overexpressing PfA-M17 displaying resistance to this drug, suggesting PfA-M17 is its target in vivo (Gardiner et al., 2006; Nankya-Kitaka et al., 1998). Harbut et al., 2011 also showed that treatment of P. falciparum with bestatin reduced hemoglobin digestion and decreased isoleucine uptake. However, specific inhibition of PfA-M17 using an activity-based probe based on the bestatin scaffold resulted in ring-stage arrest and parasite death, whilst an equivalent probe designed to specifically inhibit PfA-M1 resulted in DV swelling and stalling of parasite growth much later at the trophozoite stage. This led the authors to conclude that PfA-M17 may be playing a role outside of, or in addition to, hemoglobin digestion (Harbut et al., 2011). Several series of inhibitors designed to inhibit both PfA-M17 and PfA-M1 have also been developed and these suppress a range of Plasmodium species in vivo and in vitro (Mistry et al., 2014; Skinner-Adams et al., 2012; Vinh et al., 2019). Drinkwater et al., 2016, for example, developed a series of dual inhibitors that killed sensitive and multi-drug resistant parasites in the micromolar range, validating PfA-M1 and PfA-M17 as potential novel therapeutic targets.

PfA-M17 is a 68 kDa cytoplasmic enzyme that forms a homo-hexamer in its active form, with optimal function at neutral pH, similar to the pH of the parasite cytoplasm (Dalal and Klemba, 2007; Mathew et al., 2021; McGowan et al., 2010). Mathew et al., 2021 recently confirmed the cytoplasmic localization of PfA-M17, which suggests that hemoglobin-derived peptides are exported from the DV into the parasite cytoplasm, either through the chloroquine-resistance transporter or by other unidentified mechanisms (Shafik et al., 2020). Once in the cytoplasm, it is believed that hemoglobin-derived peptides are digested by PfA-M17; however, it is also possible that PfA-M17 plays an additional role in the catabolic turnover of peptides from other origins. Both PfA-M17 and PfA-M1 are able to cleave single amino acids from the N-terminus of small peptide chains, with PfA-M1 showing a broad substrate specificity with a preference for Met >Ala > Leu over the charged and/or polar residues Lys >Arg >> Gln (Poreba et al., 2012). In contrast, PfA-M17 shows almost exclusive selectivity for leucine and tryptophan in vitro, with leucine being one of the most abundant amino acids in adult hemoglobin (Hill et al., 1962; Poreba et al., 2012). Functionally, leucine has been shown to be an important substrate of the isoleucine transporter at the parasite membrane (Martin and Kirk, 2007). As isoleucine is the only amino acid that is absent from hemoglobin and is sourced from the host serum, leucine generated by the parasite may be important for the uptake of isoleucine across the parasite membrane (Babbitt et al., 2012; Liu et al., 2006). While repeated attempts to knockout Pfa-m17 have failed (Dalal and Klemba, 2007; Zhang et al., 2018), suggesting it is essential for intra-erythrocytic growth, its ortholog could be disrupted in both Plasmodium berghei, a rodent malaria species, and in the closely related apicomplexan parasite, Toxoplasma gondii, leading in both cases to a significant effect on parasite replication (Lin et al., 2015; Zheng et al., 2015).

In order to functionally characterize PfA-M17 and determine its contribution to P. falciparum survival, we created a conditional knockdown parasite line that enabled PfA-M17 expression to be regulated via a riboswitch (Prommana et al., 2013). This revealed that parasites depleted of PfA-M17 experience a growth delay and fail to expand in culture, subsequently leading to parasite death. We have also designed, synthesized, and characterized a novel and specific small molecule inhibitor to PfA-M17, compound 3, and parasites treated with this inhibitor demonstrate a similar growth phenotype to PfA-M17 knockdown parasites. We also found that loss of PfA-M17, either by knockdown or treatment with 3, resulted in multiple hemozoin fragments that were representative of multiple digestive vacuoles within trophozoite stage parasites. Finally, metabolomic analysis of knockdown and 3-treated parasites revealed that peptides accumulating after the depletion of PfA-M17 are likely to originate from hemoglobin, indicating that PfA-M17 plays a role in the final stages of its digestion.

For many years, PfA-M17 has been implicated in the final stage of hemoglobin digestion without any definitive confirmation. Here, using a well-established conditional system to knockdown expression of PfA-M17, we demonstrate that specific loss of this protein leads to delayed growth and eventual stalling of parasite development upon transition into trophozoite stage in the cycle following knockdown of the protein. This delay in parasite death is likely attributed to the level of knockdown occurring within the first cycle. The resulting phenotype is fatal, with parasites unable to propagate and advance through further cycles, consistent with previous conclusions that PfA-M17 is likely to be an essential protein on the basis that Pfa-m17 is refractory to gene knockout (Dalal and Klemba, 2007; Zhang et al., 2018). This validates PfA-M17 as a potential novel drug target.

Unlike previous studies on PfA-M17, which have used external activity-based probes or compounds, the conditional knockdown strategy negates any off-target effects that may have convoluted the dissection of its function. Using the approaches herein, we were additionally able to show that 3, specifically designed to target PfA-M17, was on-target using unbiased thermal proteomics profiling, and that parasites treated with this compound displayed a comparable phenotype and metabolic profile to parasites in which PfA-M17 expression had been conditionally depleted. We were unable to generate parasites resistant to 3, which is not necessarily surprising given PfA-M17 is an essential protein, and it may be impervious to mutations that permit parasite survival under the drug doses used here. Wash out experiments showed that parasites were more susceptible to 3 after they had been treated during at least one trophozoite phase, keeping with PfA-M17 appearing to be essential for this stage of growth as seen with the knockdown phenotype. Analysis of the significantly disrupted metabolites after PfA-M17 knockdown or treatment with 3 revealed an increase in abundance of peptides, many of which are likely to be derived from hemoglobin as MS/MS analysis shows the sequences of these peptides to be more similar to hemoglobin than other host or parasite proteins. This suggests that the failure to generate a sufficient pool of some amino acids from hemoglobin through a reduction in aminopeptidase activity may be the cause of parasite death. That P. falciparum parasites were significantly more sensitized to 3 when cultured in the absence of exogenous amino acids also suggests that the main function of PfA-M17 is to provide amino acids for parasite growth.

Harbut et al., 2011 previously showed that P. falciparum cultured in the presence of an activity-based probe designed to inhibit PfA-M17 resulted in parasite death at the early ring stage of the parasite lifecycle, suggesting that the role of PfA-M17 in hemoglobin digestion may be subservient to an additional but essential role it plays earlier in the lifecycle (Harbut et al., 2011). The authors hypothesized this additional role is in the turnover of peptides originating from the proteosome, as proteasome inhibitors display ring-stage killing (Prasad et al., 2013). While we cannot rule out the latter since some of the peptides that were upregulated upon PfA-M17 knockdown did not map to hemoglobin, this appears not to be the overriding function of PfA-M17 and instead the different stages at which parasites stall in the presence of the activity-based probe are likely to stem from off-target effects.

Teasing out the function of PfA-M17 has been further complicated by the fact that another aminopeptidase, PfA-M1, has also been implicated in the final stages of hemoglobin digestion. Initial studies localized PfA-M1 to the digestive vacuole, leading to speculation that the two aminopeptidases play a similar role in different parasite compartments (Dalal and Klemba, 2007). However, it is now clear that PfA-M1 is also cytoplasmic (Mathew et al., 2021). Interestingly, inhibition of PfA-M1 using an affinity-based probe resulted in parasites stalling at the trophozoite stage (Harbut et al., 2011), not dissimilar to the phenotypes observed herein upon knockdown of PfA-M17 or treatment with 3. However, treatment with this PfA-M1 affinity-based probe also resulted in swelling of the digestive vacuole, a phenotype that we did not observe with PfA-M17 knockdown; this phenotype has previously been seen when enzymes within the digestive vacuole implicated at the earlier stages of hemoglobin digestion have been targeted by compounds (Harbut et al., 2011; Rosenthal et al., 1988). Given that PfA-M17 and PfA-M1 are both cytoplasmic proteins, this then begs the question whether there is redundancy between the two, as has previously been shown with other enzymes involved in hemoglobin digestion such as falcipains and plasmepsins (Liu et al., 2006). Our results, however, indicate that loss of PfA-M17 cannot be compensated for and as Pfa-m1 has also been previously shown to be impervious to gene knockout, this would suggest that both aminopeptidases are required for parasite survival (Dalal and Klemba, 2007; Zhang et al., 2018). A thorough analysis of the P1 substrate specificities of recombinant PfA-M17 and PfA-M1 gives some indication as to why this may be. PfA-M1 is a monomeric clan MA alanyl aminopeptidase with broad substrate specificity (Poreba et al., 2012). In contrast, PfA-M17 is a clan MF leucine aminopeptidase with a far narrower specificity for the hydrophobic amino acids with a strong preference for a P1 leucine and tryptophan residue (Poreba et al., 2012). The inhibitor 3 takes advantage of this specificity with a biphenyl ring system bound in the S1 pocket satisfying the hydrophobic preferences of PfA-M17 but with an addition of a hydroxymethyl group to allow the inhibitor to reach beyond the S1 pocket and pick up an interaction with the S-atom of Met392 (Figure 4—figure supplement 1). Taken together, the inhibitor specificity combined with the P1 substrate preference for leucine suggests PfA-M17 may be a dedicated aminopeptidase for processing leucine, with leucine being the most abundant amino acid in human hemoglobin (Hill et al., 1962). Investigation of peptides identified in our metabolomic experiments, however, does not reveal an abundance of leucine-containing peptides, a result that is unexpected. The substrate specificity analysis performed in vitro is specific but cannot account for pathway dynamics or how the enzyme functions in a biological system and this must be taken into consideration. Using a conditional knockdown system negates any potential off-target effects and so the identified peptide sequences are indeed specific to the loss of PfA-M17. As there is also aberrant DV formation after depletion of PfA-M17, it is possible that there is an unidentified feedback mechanism that affects hemoglobin digestion upstream of PfA-M17 in a manner that does not increase the quantity of hemozoin produced, leading to the diverse peptides that were observed by metabolomics. Although hemozoin was observed in the multiple DVs, vacuole integrity/lack of maturation and function is also potentially impacted and could lead to perturbed hemoglobin digestion and peptide production. For example, peptides originating from hemoglobin are required to be transported out of the DV into the cytoplasm for further cleavage and this could be impacted by aberrant digestive vacuole formation. A buildup of hemoglobin-derived peptides in the cytoplasm that cannot be processed by PfA-M17 in the cytoplasm may also lead to a dysregulation of other aminopeptidases.

We have shown that selectivity of 3 for PfA-M17 over PfA-M1 is excellent against recombinant protein but cannot rule out whether there is any inhibition of PfA-M1 within the parasite, particularly as there is a significant increase in the EC₅₀ when compared to the K_i value (>300 nM compared to 18 nM). The use of 3 at high concentrations may also compound this effect, as 10 x treatments are approaching the K_i values determined against PfA-M1. However, unbiased thermal proteomics profiling using concentrations upwards of 3 x the concentration expected to inhibit recombinant PfA-M1 did not stabilize this protein, and over the different concentrations used only PfA-M17 was identified to be significantly enriched and thus the target of 3. Differences in the level of potency of an inhibitor against recombinant enzyme and parasite growth is a phenomenon that has been previously described and could be due to potential solubility problems or the ability of the compound to cross many membranes before it can interact with its target (Mills et al., 2021). This further highlights the importance of using compounds in conjunction with genetic editing techniques to confirm that phenotypes seen are not due to off-target effects. Another important consideration for compound development against PfA-M17 will be the possible off-target effects against human aminopeptidases. Currently, the human leucyl aminopeptidases are poorly characterised and the further development of PfA-M17 inhibitors as potential antimalarial therapeutics will need to take these into consideration.

Since our results indicate that PfA-M17 digests peptides derived from hemoglobin, it is not surprising that this enzyme is not essential in the closely related apicomplexan parasite, Toxoplasma gondii, which does not infect red bloods cells and therefore is not exposed to hemoglobin (Zheng et al., 2015). However, the homologous leucine aminopeptidase is also not essential in the rodent malaria species, P. berghei (Lin et al., 2015). Analysis of the PbA-M17 recombinant enzyme identified that it had a similar substrate specificity to that of PfA-M17; however, there were notable differences in substrate specificity of the PbA-M1 vs PfA-M1 enzymes, suggesting a difference in aminopeptidase substrates between the two species (Malcolm et al., 2021). Knockout of Pba-m17 did, however, result in a delay in parasite growth, although the significance of this cannot be inferred given the small sample number of two mice (Lin et al., 2015). Notably, isoleucine is present in mouse hemoglobin but absent from human hemoglobin. In P. falciparum - infected red blood cells, the influx of extracellular isoleucine can be mediated by leucine, which serves as a substrate of the isoleucine transporter at the parasite membrane (Martin and Kirk, 2007; Poreba et al., 2012). Since PfA-M17 has been demonstrated to have considerable specificity for leucine (Poreba et al., 2012) and P. berghei can source isoleucine from hemoglobin, this may explain why PfA-M17 is essential for P. falciparum survival whilst P. berghei can survive without PbA-M17.

A novel finding observed after specific depletion of PfA-M17 was the presence of multiple membrane-bound digestive vacuoles per parasite, a phenotype not previously seen with inhibition of aminopeptidases in P. falciparum. A limitation of transmission electron microscopy used here is that sections are only 2-D, but it is clear that the DV formation is severely affected after the loss of PfA-M17. It is unclear whether this is because parasites attempt to endocytose more red blood cell cytoplasm to salvage their free amino acids pool and further investigation is required in order to elucidate the mechanism of this finding. As hemoglobin digestion begins during the ring stage, it is possible that this phenotype also stems from smaller endocytic vesicles failing to fuse to create the large digestive vacuole usually visible at trophozoite stage (Abu Bakar et al., 2010). We did not see a significant increase in the amount of hemozoin produced between parasites expressing or depleted of PfA-M17, and a similar finding was observed between P. berghei wildtype and pba-m17 knockout parasites (Lin et al., 2015). This indicates that depletion or loss of leucine aminopeptidases does not result in a significant overall increase in hemoglobin digestion resulting in altered hemozoin production.

In conclusion, we have demonstrated that PfA-M17 plays an essential role in the survival of P. falciparum and is likely playing a major role in the release of amino acids originating from hemoglobin, confirming PfA-M17 as a promising target for future antimalarial drugs. Further analysis into additional metabolites found not to map to hemoglobin will possibly provide insight into their origin and clues as to additional function(s) of PfA-M17. Encouragingly, we have found that 3 is able to kill parasites in a sub-micromolar range, and we were able to confirm PfA-M17 as the compound’s target through unbiased thermal stability challenges. As this compound is most effective against the trophozoite stage, PfA-M17 is a potential attractive partner target for ring stage antimalarial drugs. Now validated, 3 also provides scope for the further analysis into the function of PfA-M17, negating problems that arise with the death phenotype of the knockdown and providing a rationale for further development of inhibitors against PfA-M17.

Source data has been provided for Figures 3, 4, 5, 6, 7, 9, 10 and Figure 3—figure supplement 2. Metabolomics data has been provided for Figures 8, 9, and Figure 8—figure supplements 1–3. Structural data has been deposited with PDB (ID 7RIE) for Figure 5 and Figure 4—figure supplement 1. The proteomics data from Figure 6 has been uploaded to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD032358. Raw metabolomic data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID (ST002106 for Exp 1, ST002107 for Exp 2, and ST002108 for Exp 3).

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Author details

1. Rebecca CS Edgar

   1. School of Medicine, Deakin University, Geelong, Australia
   2. The Institute for Mental and Physical Health and Clinical Translation, Deakin University, Geelong, Australia

   Contribution

   Conceptualization, Formal analysis, Investigation, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5974-8625

2. Ghizal Siddiqui

   Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Australia

   Contribution

   Formal analysis, Investigation, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3153-3198

3. Katheryn Hjerrild

   School of Medicine, Deakin University, Geelong, Australia

   Contribution

   Investigation

   Competing interests

   No competing interests declared

4. Tess R Malcolm

   1. Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, Australia
   2. Centre to Impact AMR, Monash University, Melbourne, Australia

   Contribution

   Investigation

   Competing interests

   No competing interests declared

5. Natalie B Vinh

   Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Australia

   Contribution

   Conceptualization, Investigation

   Competing interests

   No competing interests declared

6. Chaille T Webb

   1. Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, Australia
   2. Centre to Impact AMR, Monash University, Melbourne, Australia

   Contribution

   Formal analysis

   Competing interests

   No competing interests declared

7. Clare Holmes

   CSIRO Australian Centre for Disease Preparedness, Geelong, Australia

   Contribution

   Formal analysis, Investigation, Writing – original draft

   Competing interests

   No competing interests declared

8. Christopher A MacRaild

   Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Australia

   Contribution

   Formal analysis, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

9. Hope C Chernih

   1. School of Medicine, Deakin University, Geelong, Australia
   2. The Institute for Mental and Physical Health and Clinical Translation, Deakin University, Geelong, Australia

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2620-9518

10. Willy W Suen

    CSIRO Australian Centre for Disease Preparedness, Geelong, Australia

    Contribution

    Formal analysis, Investigation, Writing – original draft

    Competing interests

    No competing interests declared

11. Natalie A Counihan

    1. School of Medicine, Deakin University, Geelong, Australia
    2. The Institute for Mental and Physical Health and Clinical Translation, Deakin University, Geelong, Australia

    Contribution

    Formal analysis, Supervision, Investigation, Writing – review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-8973-3344

12. Darren J Creek

    Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Australia

    Contribution

    Supervision, Writing – original draft, Writing – review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-7497-7082

13. Peter J Scammells

    Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Australia

    Contribution

    Conceptualization, Supervision, Writing – original draft

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-2930-895X

14. Sheena McGowan

    1. Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, Australia
    2. Centre to Impact AMR, Monash University, Melbourne, Australia

    Contribution

    Conceptualization, Formal analysis, Supervision, Investigation, Writing – original draft, Writing – review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-6863-1106

15. Tania F de Koning-Ward

    1. School of Medicine, Deakin University, Geelong, Australia
    2. The Institute for Mental and Physical Health and Clinical Translation, Deakin University, Geelong, Australia

    Contribution

    Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Writing – original draft, Project administration, Writing – review and editing

    For correspondence

    taniad@deakin.edu.au

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-5810-8063

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