Oral Presentation Lorne Infection and Immunity 2023

Genetic and chemical validation of Plasmodium falciparum aminopeptidase PfA-M17 as an anti-malarial drug target in the haemoglobin digestion pathway (#52)

Rebecca Edgar 1 2 3 , Ghizal Siddiqui 4 , Kathryn Hjerrild 1 , Tess R Malcolm 5 , Natalie B Vinh 6 , Chaille T Webb 5 6 , Clare Holmes 7 , Christopher A MacRaild 4 , Hope C Chernih 1 2 , Willy W Suen 7 , Natalie A Counihan 1 2 , Darren J Creek 4 , Peter J Scammells 4 , Sheena McGowan 5 8 , Tania F de Koning-Ward 1 2
  1. School of Medicine, Deakin University, Waurn Ponds, Victoria, Australia
  2. The Institute for Mental and Physical Health and Clinical Translation, Deakin University, Geelong, Victoria, Australia
  3. Deakin University, St Albans Park, VIC, Australia
  4. Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, Victoria, Australia
  5. Biomedicine Discovery Institute and Department of Microbiology, , Monash University, Melbourne, Victoria, Australia
  6. Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University , Melbourne, Victoria, Australia
  7. CSIRO, CSIRO Australian Centre for Disease Preparedness, Geelong, Victoria, Australia
  8. Centre to Impact AMR, Monash University, Melbourne, Victoria, Australia

Plasmodium falciparum, a causative agent of malaria, remains a global health problem due to increasing drug resistance to therapeutics. Drugs with novel modes of action are desperately needed to combat this resistance. It is the intra-erythrocytic cycle of P. falciparum that is responsible for the clinical manifestations of disease. Here, P. falciparum infects human red blood cells and digests the cells main protein constituent, haemoglobin, in a specialised digestive vacuole. Digestion occurs in a step-wise process, with many of the enzymes in the cascade having overlapping, redundant functions. Leucine aminopeptidase PfA-M17 is one of several aminopeptidases implicated in the final step of this digestive pathway, but currently there is little evidence of its essentiality and its biological function is unconfirmed.

Here we utilised reverse genetics to generate a parasite line in which PfA-M17 can be conditionally depleted and showed that it is essential for P. falciparum survival. We additionally created a compound specifically designed to inhibit the activity of PfA-M17, which we confirmed as on target using thermal proteomics profiling and found it to kill parasites in a sub-micromolar range. Using a metabolomic approach we found that PfA-M17 provides parasites with free amino acids for growth, many of which are highly likely to originate from haemoglobin. Moreover, parasites grown in the absence of non-essential amino acids become more sensitive to our PfA-M17 inhibitor, confirming PfA-M17’s function is to provide amino acids essential for parasite survival. A further novel finding was that loss of PfA-M17 results in parasites exhibiting multiple digestive vacuoles at the trophozoite stage, which we were able to confirm via electron microscopy. In contrast to other haemoglobin-degrading proteases that have overlapping redundant functions, we validate PfA-M17 as a potential novel drug target worthy of future antimalarial development.