title: Picomolar Inhibition of Plasmepsin V, an Essential Malaria Protease, Achieved Exploiting the Prime Region.
abstract: Malaria is an infectious disease caused by Plasmodium parasites. It results in an annual death-toll of ~ 600,000. Resistance to all medications currently in use exists, and novel antimalarial drugs are urgently needed. Plasmepsin V (PmV) is an essential Plasmodium protease and a highly promising antimalarial target, which still lacks molecular characterization and drug-like inhibitors. PmV, cleaving the PExEl motif, is the key enzyme for PExEl-secretion, an indispensable parasitic process for virulence and infection. Here, we describe the accessibility of PmV catalytic pockets to inhibitors and propose a novel strategy for PmV inhibition. We also provide molecular and structural data suitable for future drug development. Using high-throughput platforms, we identified a novel scaffold that interferes with PmV in-vitro at picomolar ranges (~ 1,000-fold more active than available compounds). Via systematic replacement of P and P' regions, we assayed the physico-chemical requirements for PmV inhibition, achieving an unprecedented IC50 of ~20 pM. The hydroxyethylamine moiety, the hydrogen acceptor group in P2', the lipophilic groups upstream to P3, the arginine and other possible substitutions in position P3 proved to be critically important elements in achieving potent inhibition. In-silico analyses provided essential QSAR information and model validation. Our inhibitors act 'on-target', confirmed by cellular interference of PmV function and biochemical interaction with inhibitors. Our inhibitors are poorly performing against parasite growth, possibly due to poor stability of their peptidic component and trans-membrane permeability. The lowest IC50 for parasite growth inhibition was ~ 15 μM. Analysis of inhibitor internalization revealed important pharmacokinetic features for PExEl-based molecules. Our work disclosed novel pursuable drug design strategies for highly efficient PmV inhibition highlighting novel molecular elements necessary for picomolar activity against PmV. All the presented data are discussed in respect to human aspartic proteases and previously reported inhibitors, highlighting differences and proposing new strategies for drug development.
authors.affiliation: Department of Pharmaceutical Sciences, Università degli Studi di Milano, Milan, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Gambini
authors.affiliation: Department of Pharmaceutical Sciences, Università degli Studi di Milano, Milan, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Rizzi
authors.affiliation: Department of Pharmaceutical Sciences, Università degli Studi di Milano, Milan, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Pedretti
authors.affiliation: Department of Chemistry of Natural Substances, Faculty of Pharmacy, Università di Napoli "Federico II", Naples, Italy.
authors.forename: Orazio
authors.initials: O
authors.lastname: Taglialatela-Scafati
authors.affiliation: Department of Experimental Medicine and Biochemical Sciences, Università degli Studi di Perugia, Perugia, Italy.
authors.forename: Mario
authors.initials: M
authors.lastname: Carucci
authors.affiliation: Department of Pharmaceutical Sciences, Università degli Studi di Milano, Milan, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Pancotti
authors.affiliation: Department of Pharmaceutical Sciences, Università degli Studi di Milano, Milan, Italy.
authors.forename: Corinna
authors.initials: C
authors.lastname: Galli
authors.affiliation: Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.
authors.forename: Martin
authors.initials: M
authors.lastname: Read
authors.affiliation: Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.
authors.forename: Emanuele
authors.initials: E
authors.lastname: Giurisato
authors.affiliation: Department of Pharmaceutical Sciences, Università degli Studi di Milano, Milan, Italy.
authors.forename: Sergio
authors.initials: S
authors.lastname: Romeo
authors.affiliation: Department of Experimental Medicine and Biochemical Sciences, Università degli Studi di Perugia, Perugia, Italy.|Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Russo
mesh_terms: D000962:Antimalarials / Q000737:chemistry / Q000494:pharmacology*; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain / Q000187:drug effects; D006801:Humans; D016778:Malaria, Falciparum / Q000188:drug therapy* / Q000469:parasitology; D062105:Molecular Docking Simulation; D010963:Plasmodium falciparum / Q000737:chemistry / Q000187:drug effects* / Q000201:enzymology / Q000254:growth & development; D011480:Protease Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism
chemical_list: D000962:Antimalarials; D011480:Protease Inhibitors; D015800:Protozoan Proteins; D016282:Aspartic Acid Endopeptidases; C089643:plasmepsin