Chemoproteomics validates selective targeting of Plasmodium M1 alanyl aminopeptidase as an antimalarial strategy

  1. Darren Creek
  2. Carlo Giannangelo
  3. Matthew Challis
  4. Ghizal Siddiqui
  5. Rebecca Edgar
  6. Tess Malcolm
  7. Chaille Webb
  8. Nyssa Drinkwater
  9. Natalie Vinh
  10. Christopher MacRaild
  11. Natalie Counihan
  12. Sandra Duffy
  13. Sergio Wittlin
  14. Shane Devine
  15. Vicky Avery
  16. Tania de Koning-Ward
  17. Peter Scammells
  18. Sheena McGowan

  • Curated by eLife

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    eLife assessment

    The manuscript constitutes an important contribution to antimalarial drug discovery, employing diverse systems biology methodologies; with a focus on an improved M1 metalloprotease inhibitor, the study provides convincing evidence of the utility of chemoproteomics in elucidating the preferential targeting of PfA-M1. Additionally, metabolomic analysis effectively documents specific alterations in the final steps of hemoglobin breakdown. These findings underscore the potential of the developed methodology, not only in understanding PfA-M1 targeting but also in its broader applicability to diverse malarial proteins or pathways. Revisions are needed to further enhance overall clarity and detail the scope of these implications.

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Abstract

New antimalarial drug candidates that act via novel mechanisms are urgently needed to combat malaria drug resistance. Here, we describe the multi-omic chemical validation of Plasmodium M1 alanyl metalloaminopeptidase as an attractive drug target using the selective inhibitor, MIPS2673. MIPS2673 demonstrated potent inhibition of recombinant Plasmodium falciparum ( Pf A-M1) and Plasmodium vivax ( Pv A-M1) M1 metalloaminopeptidases, with selectivity over other Plasmodium and human aminopeptidases, and displayed excellent in vitro antimalarial activity with no significant host cytotoxicity. Orthogonal label-free chemoproteomic methods based on thermal stability and limited proteolysis of whole parasite lysates revealed that MIPS2673 solely targets Pf A-M1 in parasites, with limited proteolysis also enabling estimation of the binding site on Pf A-M1 to within ~5 Å of that determined by X-ray crystallography. Finally, functional investigation by untargeted metabolomics demonstrated that MIPS2673 inhibits the key role of Pf A-M1 in haemoglobin digestion. Combined, our unbiased multi-omic target deconvolution methods confirmed the on-target activity of MIPS2673, and validated selective inhibition of M1 alanyl metalloaminopeptidase as a promising antimalarial strategy.

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  1. Version published to 10.21203/rs.3.rs-3251230/v3 on Research Square
  2. Version published to 10.21203/rs.3.rs-3251230/v2 on Research Square
  3. eLife

    eLife assessment

    The manuscript constitutes an important contribution to antimalarial drug discovery, employing diverse systems biology methodologies; with a focus on an improved M1 metalloprotease inhibitor, the study provides convincing evidence of the utility of chemoproteomics in elucidating the preferential targeting of PfA-M1. Additionally, metabolomic analysis effectively documents specific alterations in the final steps of hemoglobin breakdown. These findings underscore the potential of the developed methodology, not only in understanding PfA-M1 targeting but also in its broader applicability to diverse malarial proteins or pathways. Revisions are needed to further enhance overall clarity and detail the scope of these implications.

  4. eLife

    Reviewer #1 (Public Review):

    Summary:
    The article "Chemoproteomics validates selective targeting of Plasmodium M1 alanyl aminopeptidase as a cross-species strategy to treat malaria" presents a series of biochemical methods based on proteomics and metabolomics, as a means to:
    (1) validate the specific targeting of biologically active molecules (MIPS2673) towards a defined (unique) protein target within a parasite
    and
    (2) to explore whether by quantifying the perturbations generated at the level of the parasite metabolome, it is possible to extrapolate which metabolic pathway has been disrupted by using this biologically active molecule and whether this may further confirm selective targeting in parasites of the expected (or in-vitro targeted) enzyme (here PfA-1).

    The inhibitor used in this work by the authors (MIPS2673) is to my knowledge a novel one, although belonging to a chemical series previously explored by the authors, which recently enabled them to discover a specific PfA-M17 inhibitor, MIPS2571 (Edgard et al., 2022, ref 11 of this current work). Indeed, inhibitors specifically targeting either PfA-M1 or PfA-M17 (and not both, as currently done in the past) are scarce today, and highly needed to functionally characterize these two zinc-aminopeptidases. MIPS2673, blocks the development of erythrocytic stages of Plasmodium falciparum with an EC50 of 324 nM, blocks the parasite development at the young trophozoite stage at 5x EC50 (but at ring stages at 10xEC50, figure 1E), and inhibits the enzymatic activity of PfA-M1 (and its ortholog Pv-M1) but not of the related malarial metallo-aminopeptidases (M17 and M18 families) nor the human metalloenzymes from closely related enzymatic families, supporting its selective targeting of PfA-M1 (and Pv-M1).

    All experiments are carried out in vitro (e.g. biochemical studies such as enzymology, proteomics, metabolomics) and on cultured parasites (erythrocyte stages of Plasmodium falciparum and several gametocytes stages obtained in vitro); there are no in vivo manipulations. The work related to Plasmodium vivax, which justifies the "cross-species" indication in the title of the article, is restricted to using a recombinant form of the M1-family aminopeptidase in enzymatic assays. The rest of the work concerns only Plasmodium falciparum. While I found globally that this work is original and brings new data and above all proposes chemical validation approaches that could be used for other target validations under similar limiting conditions (impossibility of KO of the gene), I have some specific questions to address to the authors.

    Strengths and weaknesses:
    -The chemoproteomic approach, that explores the ability of MIPS2673 to more significantly "protect" the putative target (PfA-M1) against thermal degradation or enzymatic attack (by proteinase K), to document its selective targeting towards PfA-M1 (the inhibitor, once associated with its target, is expected to stabilize its structure or prevent the action of end proteases), uses several concentrations of MIPS2673 and provides convincing results. My main criticism is that these tests are carried out with parasite extracts enriched in 30-38 hours old forms, and restricted to the fraction of soluble proteins isolated from these parasitic forms, which still limits the scope of the analysis. It is clear that this methodological approach is a choice that can be argued both biologically (PfA-M1 is well expressed in these stages of the parasite development) and biochemically (it is difficult to do proteomic analyses on insoluble proteins) but I regret that the authors do not discuss these limitations further, notably, I would have expected (from Figure 1E) some targets to be also present at ring stages.

    -The metabolomic approach, by documenting the ability of MIPS2673 to selectively increase the number of non-hydrolyzed dipeptides in treated versus untreated parasites is another argument in favor of the selective targeting of PfA-M1 by MIPS2673, in particular by its broad-spectrum aminopeptidase action preferentially targeting peptides resulting from the degradation of hemoglobin by the parasite. The relative contribution of peptides derived from host hemoglobin versus other parasite proteins is, however, little discussed.

    The work as a whole remains highly interesting, both for the specific topic of PfA-M1's role in parasite biology and for the method, applicable to other malarial drug contexts.

  5. eLife

    Reviewer #2 (Public Review):

    In this manuscript, the authors first developed a new small molecular inhibitor that could target specifically the M1 metalloproteases of both important malaria parasite species Plasmodium falciparum and P. vivax. This was done by a chemical modification of a previously developed molecule that targets PfM1 as well as PfM17 and possibly other Plasmodial metalloproteases. After the successful chemical synthesis, the authors showed that the derived inhibitor, named MIPS2673, has a strong antiparasitic activity with IC50 342 nM and it is highly specific for M1. With this in mind, the authors first carried out two large-scale proteomics to confirm the MIPS2673 interaction with PfM1 in the context of the total P. falciparum protein lysate. This was done first by using thermal shift profiling and subsequently limited proteolysis. While the first demonstrated overall interaction, the latter (limited proteolysis) could map more specifically the site of MIPS2673-PfM1 interaction, presumably the active site. Subsequent metabolomics analysis showed that MIPS2673 cytotoxic inhibitory effect leads to the accumulation of short peptides many of which originate from hemoglobin. Based on that the authors argue that the MIPS2673 mode of action (MOA) involves inhibition of hemoglobin digestion that in turn inhibits the parasite growth and development.

  6. eLife

    Reviewer #3 (Public Review):

    This is a manuscript that attempts to validate Plasmodium M1 alanyl aminopeptidase as a target for antimalarial drug development. The authors provide evidence that MIPS2673 inhibits recombinant enzymes from both Pf and Pv and is selective over other proteases. There is in vitro antimalarial activity. Chemoproteomic experiments demonstrate selective targeting of the PfA-M1 protease.

    This is a continuation of previous work focused on designing inhibitors for aminopeptidases by a subset of these authors. Medicinal chemistry explorations resulted in the synthesis of MIPS2673 which has improved properties including potent inhibition of PfA-M1 and PvA-M1 with selectivity over a closed related peptidase. The compound also demonstrated selectivity over several human aminopeptidases and was not toxic to HEK293 cells at 40 uM. The activity against P. falciparum blood-stage parasites was about 300 nM.

    Thermal stability studies confirmed that PfA-M1 was a binding target, however, there were other proteins consistently identified in the thermal stability studies. This raises the question as to their potential role as additional targets of this inhibitor. The authors dismiss these because they are not metalloproteases, but further analysis is warranted. This is particularly important as the authors were not able to generate mutants using in vitro evolution of resistance strategies. This often indicates that the inhibitor has more than one target.

    The next set of experiments focused on a limited proteolysis approach. Again several proteins were identified as interacting with MIPS2673 including metalloproteases. The authors go on to analyze the LiP-MS data to identify the peptide from PfA-M1 which putatively interacts with MIPS2673. The authors are clearly focused on PfA-M1 as the target, but a further analysis of the other proteins identified by this method would be warranted and would provide evidence to either support or refute the authors' conclusions.

    The final set of experiments was an untargeted metabolomics analysis. They identified 97 peptides as significantly dysregulated after MIPS2673 treatment of infected cells and most of these peptides were derived from one of the hemoglobin chains. The accumulation of peptides was consistent with a block in hemoglobin digestion. This experiment does reveal a potential functional confirmation, but questions remain as to specificity.

    Overall, this is an interesting series of experiments that have identified a putative inhibitor of PfA-M1 and PvA-M1. The work would be significantly strengthened by structure-aided analysis. It is unclear why putative binding sites cannot be analyzed via specific mutagenesis of the recombinant enzyme. In the thermal stability and LiP -MS analysis, other proteins were consistently identified in addition to PfA-M1 and yet no additional analysis was undertaken to explore these as potential targets. The metabolomics experiments were potentially interesting, but without significant additional work including different lengths of treatment and different stages of the parasite, the conclusions drawn are overstated. Many treatments disrupt hemoglobin digestion - either directly or indirectly and from the data presented here it is premature to conclude that treatment with MIPS2673 directly inhibits hemoglobin digestion. Finally, the potency of this compound on parasites grown in vitro is 300 nM - this would need improvements in potency and demonstration of in vivo efficacy in the SCID mouse model to consider this a candidate for a drug.

    Summary:
    Overall, this is an interesting series of experiments that have identified a putative inhibitor of the Plasmodium M1 alanyl aminopeptidases, PfA-M1 and PvA-M1.

    Strengths:
    The main strengths include the synthesis of MIPS2673 which is selectively active against the enzymes and in whole-cell assay.

    Weaknesses:
    The weaknesses include the lack of additional analysis of additional targets identified in the chemoproteomic approaches.

  7. Version published to 10.21203/rs.3.rs-3251230/v1 on Research Square