**4. Conclusions**

Molecular docking is one of the most widely used computational approaches in the design of structure-based drugs [67,68]. This approach can be used both to identify the correct conformation of the ligand within the target binding pocket and to estimate the interaction energy between a target and a ligand [69,70]. In the present study, different Syk inhibitors were investigated using different methodologies to test their efficacy in vitro and in silico as promising new antimalarial drugs to block Syk phosphorylation events in RBCs infected by parasites. Thorough the computational studies, we gathered useful information about the tested inhibitors, analyzing their chemical features and the

patterns of interaction in the Syk protein pocket, proving their inhibitory activity in in vitro experiments. Based on the above data and other publications from our labs, we demonstrated that these compounds actually block the phosphorylation of Tyr residues in protein band 3. The in vitro experiments using a proteomic approach allowed us to notice that Syk inhibitors terminate the life cycle during the process of merozoite egress from the infected RBC, leading to a decrease of band 3 phosphorylation and avoiding the destabilization and weakening of the erythrocyte membrane. The biological data obtained by evaluating the IC50 values using Western blotting for all tested compounds confirmed the results achieved in in silico studies. The development of new drugs that can effectively kill malaria parasites is of increasing importance as the threat of drug resistance continues to grow. However, as highlighted above, the findings of this study require further exploration and testing to elucidate or confirm the role of Syk inhibitors as potential drug targets.

**Author Contributions:** A.P., G.M., A.D., and R.D. conceived of the study and design and supervised the study, contributing to interpretation of data and writing of the paper. G.M., together with I.T., A.D., and R.D., performed experiments. M.C.P. and I.T. contributed to the formulation of results. A.P., F.M.T., A.D., and R.D. have given approval to the final version of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by grants from Fondo di Ateneo per la Ricerca 2020.

**Acknowledgments:** The authors would like to thank Claudio Fozza for his collaboration.

**Conflicts of Interest:** The authors declare no conflict of interest.
