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

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

  1. Carlo Giannangelo
  2. Matthew P Challis
  3. Ghizal Siddiqui
  4. Rebecca Edgar
  5. Tess R Malcolm
  6. Chaille T Webb
  7. Nyssa Drinkwater
  8. Natalie Vinh
  9. Christopher Macraild
  10. Natalie Counihan
  11. Sandra Duffy
  12. Sergio Wittlin
  13. Shane M Devine
  14. Vicky M Avery
  15. Tania De Koning-Ward
  16. Peter Scammells
  17. Sheena McGowan
  18. Darren J Creek  Is a corresponding author
  1. Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Australia
  2. School of Medicine, Deakin University, Australia
  3. The Institute for Mental and Physical Health and Clinical Translation, Deakin University, Australia
  4. Monash Biomedicine Discovery Institute and Department of Microbiology, Monash University, Australia
  5. Centre to Impact AMR, Monash University, Australia
  6. Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Australia
  7. Discovery Biology, Centre for Cellular Phenomics, Griffith University, Australia
  8. Swiss Tropical and Public Health Institute, Switzerland
  9. University of Basel, Switzerland
  10. The Walter and Eliza Hall Institute of Medical Research, Australia
  11. Department of Medical Biology, The University of Melbourne, Australia
  12. School of Environment and Science, Griffith University, Australia
https://doi.org/10.7554/eLife.92990.3
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Cite this article
  1. Carlo Giannangelo
  2. Matthew P Challis
  3. Ghizal Siddiqui
  4. Rebecca Edgar
  5. Tess R Malcolm
  6. Chaille T Webb
  7. Nyssa Drinkwater
  8. Natalie Vinh
  9. Christopher Macraild
  10. Natalie Counihan
  11. Sandra Duffy
  12. Sergio Wittlin
  13. Shane M Devine
  14. Vicky M Avery
  15. Tania De Koning-Ward
  16. Peter Scammells
  17. Sheena McGowan
  18. Darren J Creek
(2024)
Chemoproteomics validates selective targeting of Plasmodium M1 alanyl aminopeptidase as an antimalarial strategy
eLife 13:RP92990.
https://doi.org/10.7554/eLife.92990.3
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6 figures, 4 tables and 1 additional file

Figures

Figure 1 with 1 supplement
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Synthesis and antimalarial activity of the M1-selective aminopeptidase inhibitor, MIPS2673.

(A) Synthesis of MIPS2673 (4). Reagents and conditions: (i) RCOOH, EDCI, DMAP, CH2Cl2; (ii) 3,4,5-trifluorophenylboronic acid, Pd(PPh3)2Cl2, Na2CO3, THF; (iii) NH2OH•HCl, KOH. (B) Binding mode of …

Figure 1—source data 1

Data collection and refinement statistics for X-ray crystal structure.

https://cdn.elifesciences.org/articles/92990/elife-92990-fig1-data1-v1.docx
Figure 1—figure supplement 1
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Antimalarial potency of the clinically used artemisinin derivative, artesunate.

Sensitivity to artesunate exposure (72 hr) for P. falciparum 3D7 parasites. Artesunate activity was tested alongside MIPS2673 as a reference compound with known antimalarial activity. Parasite …

Figure 2
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Experimental thermal stability and limited proteolysis workflows for unbiased drug target identification in P. falciparum lysates.

Synchronised trophozoite-stage parasites (30–38 hr post invasion) are isolated from the host red blood cell by hypotonic lysis. Proteins are extracted from cells under native conditions by multiple …

Figure 3 with 1 supplement
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Thermal stabilisation of proteins by MIPS2673.

(A) Paired volcano plots of all proteins detected after a 60°C thermal challenge across two experiments from P. falciparum lysates (at least n=3 of independent lysate incubations per condition and …

Figure 3—source data 1

Thermal stability proteomics datasets related to Figure 3.

https://cdn.elifesciences.org/articles/92990/elife-92990-fig3-data1-v1.xlsx
Figure 3—source data 2

Thermal stability proteomics joint volcano plot data related to Figure 3.

https://cdn.elifesciences.org/articles/92990/elife-92990-fig3-data2-v1.xlsx
Figure 3—figure supplement 1
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Individual thermal stabilisation profile of PfA-M17 leucyl aminopeptidase after P. falciparum lysate treatment with MIPS2673.

Abundance of PfA-M17 leucyl aminopeptidase from P. falciparum lysates in the absence and presence of MIPS2673 after a 60°C thermal challenge. PfA-M17 was not stabilised by MIPS2673 after a 60°C …

Figure 4 with 1 supplement
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Identification of MIPS2673 target proteins using limited proteolysis coupled with mass spectrometry (LiP-MS).

(A) Principle of LiP-MS analysis. Ligand binding to a protein alters the local proteolytic susceptibility and prevents protein cleavage by proteinase K. In the bound state this results in decreased …

Figure 4—source data 1

Limited proteolysis coupled with mass spectrometry (LiP-MS) proteomics datasets related to Figure 4 and Figure 5.

https://cdn.elifesciences.org/articles/92990/elife-92990-fig4-data1-v1.xlsx
Figure 4—source data 2

Limited proteolysis coupled with mass spectrometry (LiP-MS) Fisher’s exact test p-values related to Figure 4.

https://cdn.elifesciences.org/articles/92990/elife-92990-fig4-data2-v1.xlsx
Figure 4—source data 3

Experiment one: limited proteolysis coupled with mass spectrometry (LiP-MS) gene ontology (GO) molecular function enrichment analysis related to Figure 4.

https://cdn.elifesciences.org/articles/92990/elife-92990-fig4-data3-v1.xlsx
Figure 4—figure supplement 1
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Molecular function gene ontology (GO) enrichment analysis of the MIPS2673 experiment one: limited proteolysis coupled with mass spectrometry (LiP-MS) dataset.

Fold enrichment (left) and significance (right) of GO molecular functions enriched among the expanded list of candidate MIPS2673 targets identified in the first LiP-MS experiment. All proteins with …

Figure 5 with 2 supplements
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Features of structurally significant PfA-M1 limited proteolysis coupled with mass spectrometry (LiP-MS) peptides.

(A) Relative abundance of the significant LiP peptides commonly identified across two LiP-MS experiments following P. falciparum proteome lysate treatment with MIPS2673. The mean ± SEM of at least …

Figure 5—figure supplement 1
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Minimum distance of significant limited proteolysis coupled with mass spectrometry (LiP-MS) or all other PfA-M1 peptides from bound MIPS2673.

Among the 108 PfA-M1 peptides commonly identified across both LiP-MS experiments, nine peptides were significantly dysregulated in the presence of MIPS2673 at all concentrations tested. The atoms of …

Figure 5—figure supplement 2
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Minimum distance measurements and mapping of significant limited proteolysis coupled with mass spectrometry (LiP-MS) peptides to structures of other putative MIPS2673-interacting proteins.

Distance measurement analyses were performed for other putative MIPS2673-interacting proteins identified with thermal stability proteomics and LiP-MS (experiment one) where a protein structure was …

Figure 6 with 1 supplement
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Targeted analysis of significantly dysregulated peptides (p<0.05) following treatment with 1 µM of MIPS2673 (PfA-M1 inhibitor) for 1 hr compared to MIPS2571 (PfA-M17 inhibitor) and DMSO control.

(A) Hierarchical clustering of the 97 peptides significantly dysregulated after treatment with MIPS2673 (fold-change >1.5 and p<0.05); four biological replicates for MIPS2673 and MIPS2571 (data from …

Figure 6—figure supplement 1
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Untargeted metabolomics analysis of 3D7 parasites treated with 1 µM of MIPS2673.

Heatmap analysis of peak intensities of all putative metabolites for each condition; 1 µM of MIPS2673 for 1 hr and DMSO control (untreated). Data is shown from four to nine biological replicates; …

Tables

Table 1
Percent inhibition by MIPS2673 of PfA-M1 and PvA-M1 aminopeptidase activities compared to selected human M1 homologues.
Concentration (µM)% inhibition*
PfA-M1PvA-M1LTA4HERAP1ERAP2
1.251001000031
10100100582.30
500––42.289.897.8
1000––53.495.698.7
  1. *

    Values represent the mean of three independent experiments. All assays were performed at pH 8.0. The concentration of substrate H-Leu-NHMec (H-Leu-NHMec, L-leucine-7-amido-4-methylcoumarin hydrochloride) varied depending on the enzyme assayed 20 µM for PfA-M1, 40 µM for PvA-M1, and 100 µM for ERAP1 and ERAP2. LTA4H was assessed using the substrate H-Ala-NHMec (L-alanine-7-amido-4-methylcoumarin hydrochloride) at a concentration of 20 µM. LTA4H, leukotriene A-4 hydrolase. ERAP1, endoplasmic reticulum aminopeptidase 1. ERAP2, endoplasmic reticulum aminopeptidase 2.

Table 2
Cytotoxicity of MIPS2673 against the human HEK293 cell line.
Compound% inhibition*
40 µM120 µM
MIPS2673NI89
Puromycin†100103
Chloroquine6765
DHA2932
  1. *

    Values represent the mean from two experiments.

  2. †

    Puromycin was tested at 10 µM. NI, no inhibition.

Table 3
MIPS2673 effectiveness against a panel of drug-resistant P. falciparum strains*.
P. falciparum strainSensitivity profileEC50 (nM)†Fold shift in EC50 relative to
NF54Dd2
NF54Sensitive2371.0
K1Decreased sensitivity to chloroquine2781.2
7G8Decreased sensitivity to chloroquine2931.2
TM90C2BDecreased sensitivity to atovaquone1940.8
Cam3.1Decreased sensitivity to artemisinin2441.0
Dd2Decreased sensitivity to chloroquine2200.91.0
Dd2 DDD107498Decreased sensitivity to eEF2 inhibitor, DDD1074982111.0
Dd2 MMV390048Decreased sensitivity to PI4K inhibitor, MMV3900481640.7
Dd2 DSM265Decreased sensitivity to DHODH inhibitor, DSM2651830.8
Dd2 GNF156Decreased sensitivity to GNF1561950.9
Dd2 ELQ300Decreased sensitivity to cyt bc1 inhibitor, ELQ3001740.8
  1. *

    72 hr [3H]-hypoxanthine incorporation assay.

  2. †

    Mean values from two biological replicates.

Table 4
MIPS2673 effectiveness against P. falciparum early (I-III), late (IV-V), and mature (V) gametocyte stages of NF54-pfs16-GFP parasites.
CompoundEC50 (nM)*
Early-stage gametocytesLate-stage gametocytesMature-stage gametocytes
MIPS26731660±715300±192398% (120 µM)†
Artesunate16±5.026±2.051±23
  1. *

    EC50 values were determined in technical duplicate from two to three biological repeats.

  2. †

    % inhibition at 120 µM (EC50 not determined).

Additional files

MDAR checklist
https://cdn.elifesciences.org/articles/92990/elife-92990-mdarchecklist1-v1.docx

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