Periodic Reporting for period 1 - SUBUN (Engineering substrate-based inhibitors of Plasmodium SUB1, a potential target for Malaria treatment.)
Período documentado: 2020-06-01 hasta 2022-05-31
Malaria is the deadliest human parasitic disease and the spreading of multi-resistant parasites seriously threatens its treatment and control. It occurs in tropical and subtropical ecosystems of more than 100 countries. Thus, about half of the world population is at risk, while the increase in human mobility (travel and migration) and climate change are causing a rise up of illness cases outside the endemic occurrence areas. The constant selection and spreading of Plasmodium parasites (falciparum and vivax, mainly) resistant to current treatments strengthens the constant need to develop antimalarials candidates active on new targets, expressed at essential stages of Plasmodium. The egress of parasite from infected host cells is such a crucial step that strictly depends upon parasite proteases, including a novel highly promising target, the Plasmodium-specific subtilisin SUB1, for which no useful inhibitor has been discovered yet. SUB1 plays a key role in the egress from hepatocytes and erythrocytes, including for male gametes (involved in parasite’s transmission via the Anopheles vector), indicating that an inhibitor would have both prophylactic and therapeutic value and obey to most severe WHO’s criteria of future antimalarials, i.e. targeting all stages of parasite life cycle. Based on Hosts’ promising results, including the resolution of crystallographic structures of SUB1-inhibitor complexes, the SUBUN project aimed at identifying highly potent and selective SUB1 inhibitors, active against parasite growth in vitro and in vivo. After two years, highly potent and selective inhibitors have been characterized, but they showed moderate parasite growth inhibition in vitro and no in vivo evaluation could be performed yet.
Combining expertise in molecular design, medicinal chemistry, biochemistry, structural biology and parasitology, the SUBUN project initially concerned the engineering of original SUB1 inhibitors, following a substrate- and mechanism-based approach using SFTI-1 (Sunflower trypsin inhibitor), a natural serine protease inhibitor, as a template. SFTI-1 is a 14-residue long macrocyclic peptide with one disulphide bridge. Usual strategy is to adapt its reactive site and the binding domain around it to the targeted enzyme. Molecular modelling study was performed to help compound design. About 20 analogues were synthesized following standard solid phase synthetic methodology. The macrocyclization and disulphide bond formation steps had to be optimized. The SUB1 inhibitory potency of all compounds was assessed using an in vitro assay. Unfortunately, none significantly inhibited SUB1, and consequently the inhibition of parasite growth in culture was not observed. The likely reason is that the SFTI-1 scaffold is a too highly constrained molecule to adapt into the SUB1 binding site, making further development of this strategy uncertain. For this reason, the SFTI-1 approach was abandoned and we chose to explore another kind of substrate- and mechanism-based pseudo-peptidic inhibitors. In this series, previous work identified potent SUB1 inhibitors but with only moderate parasite growth inhibition in culture. The main reason was probably a low capacity of the compounds to cross cell membranes. In this context, our objective was to improve not only SUB1 affinity, but also cell penetration. Specific modifications were introduced, contributing to a large study of structure-activity relationships. This work led to the identification of about two-fold more potent inhibitors also showing higher parasite growth inhibition in vitro. Further effort is needed to obtain compounds with expected activity. Other experiments were performed to assess the druggability of these inhibitors. We found that they were selective against human serine proteases, suggesting a low risk of off-target effects related to the inhibition of these human enzymes. They also showed high stability in plasma and poor binding to albumins present in parasite culture medium, which excludes both phenomena as the cause of reduced in vitro activity. Finally, the crystallization of several SUB1-inhibitor complexes was attempted and one structure is being resolved. The obtained 3D structure will contribute to the structure-based design of future molecules.
Part of the results has been presented as a poster at the 22nd congress of the Groupe Français des Peptides et Protéines (GFPP) held in Port Leucate (France) from 29th May to 3rd June 2022 and at the 29th SCT Young Research Fellows meeting in Nantes (France). Finally, two manuscripts related to the study of structure-activity relationships are in preparation.
Part of the results has been presented as a poster at the 22nd congress of the Groupe Français des Peptides et Protéines (GFPP) held in Port Leucate (France) from 29th May to 3rd June 2022 and at the 29th SCT Young Research Fellows meeting in Nantes (France). Finally, two manuscripts related to the study of structure-activity relationships are in preparation.
Compared to previously published work related to the secondary series of pseudo-peptide inhibitors, where no analogue showed anti-parasitic activity in vitro, we identified SUB1 inhibitors inhibiting parasite growth with IC50 in the 10 M range. These promising results encouraged us to further develop this series of compounds toward the identification of a lead SUB1 inhibitor with desired in vitro and in vivo activities.
The activities carried out under the project were basic research, which is still far from having a socio-economic impact. However, due to the worldwide problem of malaria and its increasing drug resistance, such projects respond to social needs, and progress made during SUBUN in developing new drugs, which are supposed to have both healing and preventive effects, may in the future contribute to limiting the problem of high global mortality among malaria infected patients, especially in poor countries.
The activities carried out under the project were basic research, which is still far from having a socio-economic impact. However, due to the worldwide problem of malaria and its increasing drug resistance, such projects respond to social needs, and progress made during SUBUN in developing new drugs, which are supposed to have both healing and preventive effects, may in the future contribute to limiting the problem of high global mortality among malaria infected patients, especially in poor countries.