Non-haemoglobin degrading Plasmepsins: Drug targets validation from bench to field

Malaria and drug-resistant forms of malaria represent the most important parasitic disease worldwide and one of the major global threat for human health with 40% of the world’s population currently at risk, 300-500 million cases of clinical malaria and more than 1 million deaths each year affecting primarily the developing countries and children under the age of five. Plasmodium belongs to the phylum Apicomplexa and out of these five species P. falciparum is the most virulent and is responsible for almost all the deaths attributed to malaria. The dramatic extension of parasite resistance to most anti-malarial drugs is directly responsible for the widespread persistence of high incidence of malaria and related morbidity in tropical areas. It is absolutely critical to develop the technology for understanding comprehensively the biology of Plasmodium and the intimate relationships with its host in order to identify and validate novel arsenal of targets for intervention. Among the attractive targets, hemoglobin degrading pathway that supplies growth and development of parasites has been the focus of attention and intensive research. During its erythrocytic cycle, Plasmodium parasites degrade most of the host cell haemoglobin and recycle the amino acids for the biosynthesis of their own proteins (Goldberg, 2005). This catabolic process that takes place in the acidic food vacuole of the parasite involves a number of proteases: aspartic proteases (Plasmepsins I, II, IV and HAP), cysteine proteases (Falcipains) and metalloprotease (falcilicin) (Liu et al., 2006). While the haemoglobin degrading Plasmepsins are dispensable for parasite survival, Plasmepsins IX and X that localize to the Mauer’s clefts and to the apical tip of schizonts respectively are essential for parasite survival. Preliminary data on the functional dissection of PMIX indicate that this protease is essential for egress/invasion. Importantly very potent anti-malarial drugs raised against aspartyl proteases actually act on the late step of the erythrocytic cycle and impact on parasite egress. The best of these compounds shows an IC50 of 0.6 nM (Ciana et al., 2013). Itis hypothesized that those compounds target either PMIX or PMX or both. The proposal sets to challenge this hypothesis through this project that will aim to define, characterize and validate new molecular targets against malaria in vitro and in vivo using field isolates. This research project is articulated around four independent and complementary aims that can be performed in parallel. 1. Validation of Plasmespin IX and Plasmepsin X as candidate antimalarial drug targets:The conditional excision of PfPMIX gene demonstrates that this aspartyl protease is essential for parasite survival. The Swiss team will apply the same strategy to generate a Plasmodium falciparum mutant strain to conditionally disrupt the PfPMX gene. Both mutant strains will be phenotypically investigated in depth, in order to unravel the biological role and narrow down the time and site of action of these two proteases. 2. Sensitivity of PfPMIX and PfPMX to aspartly proteases inhibitors:While the hemoglobin degrading enzymes such as Plasmesin II have been extensively studied and characterized, nothing in known about PfPMIX and PfPMX, The Indian team will engage considerable efforts in exploring the heterologous expression systems with the objective of producing active proteases to perform a comprehensive biochemical and enzymatic characterization. Ultimately, the selectivity and sensitivity of these inhibition assays to different classes of aspartyl proteases inhibitors will be examined through enzymatic inhibition assays.3. Identification of the target (s) of hydroxy-ethyl-amine scaffolds active on late schizonts:The hydroxy-ethyl-amine scaffolds are highly potent antimalarials compounds targeting aspartyl proteases. Importantly the time of action (on late schizonts) exclude an action of haemoglobin degradation and points toward targeting PfPMIX and/ or PfPMX. To identify the drug target of these compounds, the Swiss team will i) define to precise time and mode of action by phenotyping the treated parasites and ii) use a chemistry approach to demonstrate the physical association of the lead compounds with aspartyl proeases in the parasites (in collaboration with Dr. R. Pradip, India/Seattle). 4. Evaluation of hydroxy-ethyl-amine scaffolds on field isolates and other malaria life stages and species: To establish if aspartyl proteases inhibitors targeting PfPMIX and/or PfPMX can be developed into clinically useful drugs for the treatment of malaria, the Indian team will determine the lead compounds based on hydroxy-ethyl-amine scaffolds on field isolates of malaria parasites including P. vivax. Taken together, this project covers a large panel of competences from the identification, functional characterization and validation of new molecular targets to the characterization of antimalarials compounds that presumably target them. Ultimately this project should integrate different technical approaches to meet the goal of supplying new drugs to improve the inadequate treatment options against malaria.

Project details

Basic Science

Apr 2015 — Jul 2018

$252,135

India
Switzerland