Synthesis-, biology- & structure-driven optimization of Plasmodium SUB1 Peptidase Inhibitors, a potential target for Malaria treatment – SPIM

Malaria, caused by Plasmodium species, particularly falciparum and vivax, is the most important human parasitic disease and the constant selection and spreading of multi-resistant parasites, including to the artemisinin-based combination therapies for falciparum, seriously threaten malaria treatment and control. After >15 years of decrease, the WHO noted the epidemy raised up again in different countries in 2017. This situation strengthens the constant need to fuel the antimalarials pipeline with candidates active on new targets expressed at essential stages of Plasmodium. The egress of merozoites from the infected host cells is such a yet untargeted essential step, which strictly depends upon parasite proteases, defining these enzymes as a new generation of potential drug targets.

We are exploring targeting SUB1, a Plasmodium-specific subtilisin protease, which is highly promising for the following reasons:
i) SUB1 is crucial for the parasite development, via playing a key dual role in both the egress of merozoites from infected hepatocytes (shown by Partner 2) and red blood cells (RBC) and the invasion into new RBC. Its role in egress from hepatocytes is important to note as the hepatic stage is the first step of the parasites after a primary infection and blocking this stage is of high preventive value. Hence, targeting SUB1 is in line with the WHO’s criteria of future anti-malarials since two different biological stages of Plasmodium life cycle would be targeted.
ii) SUB1 active site significantly differs from that of human subtilisins.
iii) Partner 2 has shown that SUB1 is well conserved in P. falciparum and P. vivax field isolates, indicating that a single drug is likely to be equally efficient against both human-infecting species. This was confirmed by the fact that inhibitors synthesized by Partner 1 were equally efficiently on both PfSUB1 and PvSUB1.
iv) Considering the high level of conservation in field isolated parasites of both SUB1 active site and its substrates, the likelihood of simultaneous compensatory mutations, and thus resistance to drug, is reduced.
v) Partner 2 has recently resolved the crystal structure of both PvSUB1 and PfSUB1, as well as that of PvSUB1 complexes with inhibitors synthesized by Partner 1, facilitating structure-based design.

Therefore, the SPIM project aims at identifying highly potent and selective inhibitors of the Plasmodium-specific subtilisin peptidase SUB1, which activity against parasite growth will be evaluated in vitro and in vivo. This project will combine expertise in molecular design, medicinal chemistry, biochemistry, structural biology and parasitology to attain this objective.

Three main tasks are defined:
1) Design and synthesis of SUB1-specific inhibitors (Partner 1): the design will be driven by structrural X-ray data and molecular modelling (Partner 2).
2) Biochemical and biological evaluations of the inhibitors (Partner 2):
a) Determination of inhibitors affinity for Plasmodium sp. Pf/Pv/PbSUB1.
b) Ex vivo evaluation of P. falciparum and P. vivax erythrocytic growth inhibition.
c) Biological correlation of SUB1 and Plasmodium merozoite egress inhibitions and cellular localization of inhibitors.
d) Evaluation of SUB1 inhibitor selectivity and non-specific toxicity.
e) In vivo anti-parasite activity on mice infected by Plasmodium sp.
f) Activity on Plasmodium hepatic merozoites egress.
3) Resolution of the binding mode to drive the design of optimized molecules: X-ray crystallography of complexes, molecular modelling (Partner 2).

Expected results are: i) Discovery of SUB1 specific inhibitor(s) inhibiting multi-resistant P. falciparum and vivax growth in vitro with IC50 below 100 nM; ii) demonstration that compounds are targeting native SUB1. iii) Proof of principle that SUB1 inhibitors are active in vivo defining pre-lead candidate(s) with innovative mode of action.