title: Binding and Inhibition of Spermidine Synthase from Plasmodium falciparum and Implications for In Vitro Inhibitor Testing.
abstract: The aminopropyltransferase spermidine synthase (SpdS) is a promising drug target in cancer and in protozoan diseases including malaria. Plasmodium falciparum SpdS (PfSpdS) transfers the aminopropyl group of decarboxylated S-adenosylmethionine (dcAdoMet) to putrescine or to spermidine to form spermidine or spermine, respectively. In an effort to understand why efficient inhibitors of PfSpdS have been elusive, the present study uses enzyme activity assays and isothermal titration calorimetry with verified or predicted inhibitors of PfSpdS to analyze the relationship between binding affinity as assessed by KD and inhibitory activity as assessed by IC50. The results show that some predicted inhibitors bind to the enzyme with high affinity but are poor inhibitors. Binding studies with PfSpdS substrates and products strongly support an ordered sequential mechanism in which the aminopropyl donor (dcAdoMet) site must be occupied before the aminopropyl acceptor (putrescine) site can be occupied. Analysis of the results also shows that the ordered sequential mechanism adequately accounts for the complex relationship between IC50 and KD and may explain the limited success of previous efforts at structure-based inhibitor design for PfSpdS. Based on PfSpdS active-site occupancy, we suggest a classification of ligands that can help to predict the KD-IC50 relations in future design of new inhibitors. The present findings may be relevant for other drug targets that follow an ordered sequential mechanism.
authors.affiliation: Center for Molecular Protein Science, Lund University, SE-221 00, Lund, Sweden.|Department of Experimental Medical Science, Lund University, SE-221 84, Lund, Sweden.
authors.forename: Janina
authors.initials: J
authors.lastname: Sprenger
authors.affiliation: Chemistry Department, Princeton University, Princeton, New Jersey, 08544, United States of America.
authors.forename: Jannette
authors.initials: J
authors.lastname: Carey
authors.affiliation: SARomics Biostructures AB, Medicon Village, SE-223 81, Lund, Sweden.
authors.forename: Bo
authors.initials: B
authors.lastname: Svensson
authors.affiliation: Center for Molecular Protein Science, Lund University, SE-221 00, Lund, Sweden.|Department of Experimental Medical Science, Lund University, SE-221 84, Lund, Sweden.
authors.forename: Verena
authors.initials: V
authors.lastname: Wengel
authors.affiliation: Department of Experimental Medical Science, Lund University, SE-221 84, Lund, Sweden.
authors.forename: Lo
authors.identifier: http://orcid.org/0000-0002-5792-6184
authors.initials: L
authors.lastname: Persson