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.
