121a4659e1a05b83edc1ce8ae66d29e5 pmc_28430158.pdf 1ac8eaf312500cdceb2844638d2897b2441a7d6f pmc_28430158.pdf a403d729d3c62969c6fe06301ab1d64550c8b320382eb735ac84373525c28c4e pmc_28430158.pdf Title: Identification of Tight-Binding Plasmepsin II and Falcipain 2 Inhibitors in Aqueous Extracts of Marine Invertebrates by the Combination of Enzymatic and Interaction-Based Assays Subject: Natural products from marine origin constitute a very promising and underexplored source of interesting compounds for modern biotechnological and pharmaceutical industries. However, their evaluation is quite challenging and requires specifically designed assays to reliably identify the compounds of interest in a highly heterogeneous and interfering context. In the present study, we describe a general strategy for the confident identification of tight-binding protease inhibitors in the aqueous extracts of 62 Cuban marine invertebrates, using Plasmodium falciparum hemoglobinases Plasmepsin II and Falcipain 2 as model enzymes. To this end, we first developed a screening strategy that combined enzymatic with interaction-based assays and then validated screening conditions using five reference extracts. Interferences were evaluated and minimized. The results from the massive screening of such extracts, the validation of several hits by a variety of interaction-based assays and the purification and functional characterization of PhPI, a multifunctional and reversible tight-binding inhibitor for Plasmepsin II and Falcipain 2 from the gorgonian Plexaura homomalla, are presented. Keywords: tight-binding protease inhibitor; combined screening strategy; Plasmepsin II; Falcipain 2; Plasmodium falciparum Author: Emir Salas-Sarduy, Yasel Guerra, Giovanni Covaleda Cortés, Francesc Xavier Avilés and María A. 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