title: Identification and characterization of allophenylnorstatine-based inhibitors of plasmepsin II, an antimalarial target.
abstract: Plasmepsin II is a key enzyme in the life cycle of the Plasmodium parasites responsible for malaria, a disease that afflicts more than 300 million individuals annually. Since plasmepsin II inhibition leads to starvation of the parasite, it has been acknowledged as an important target for the development of new antimalarials. In this paper, we identify and characterize high-affinity inhibitors of plasmepsin II based upon the allophenylnorstatine scaffold. The best compound, KNI-727, inhibits plasmepsin II with a K(i) of 70 nM and a 22-fold selectivity with respect to the highly homologous human enzyme cathepsin D. KNI-727 binds to plasmepsin II in a process favored both enthalpically and entropically. At 25 degrees C, the binding enthalpy (DeltaH) is -4.4 kcal/mol and the entropic contribution (-TDeltaS) to the Gibbs energy is -5.56 kcal/mol. Structural stability measurements of plasmepsin II were also utilized to characterize inhibitor binding. High-sensitivity differential scanning calorimetry experiments performed in the absence of inhibitors indicate that, at pH 4.0, plasmepsin II undergoes thermal denaturation at 63.3 degrees C. The structural stability of the enzyme increases with inhibitor concentration in a manner for which the binding energetics of the inhibitor can quantitatively account. The effectiveness of the best compounds in killing the malaria parasite was validated by performing cytotoxicity assays in red blood cells infected with Plasmodium falciparum. EC50s ranging between 6 and 10 microM (3-6 microg/mL) were obtained. These experiments demonstrate the viability of the allophenylnorstatine scaffold in the design of powerful and selective plasmepsin inhibitors.
authors.affiliation: Department of Biology and The Johns Hopkins Malaria Research Institute, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
authors.forename: Azin
authors.initials: A
authors.lastname: Nezami
authors.forename: Irene
authors.initials: I
authors.lastname: Luque
authors.forename: Tooru
authors.initials: T
authors.lastname: Kimura
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Kiso
authors.forename: Ernesto
authors.initials: E
authors.lastname: Freire
mesh_terms: D000577:Amides / Q000737:chemistry / Q000633:toxicity; D000818:Animals; D000962:Antimalarials / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002863:Chromogenic Compounds / Q000737:chemistry / Q000378:metabolism; D004795:Enzyme Stability / Q000187:drug effects; D004912:Erythrocytes / Q000187:drug effects / Q000201:enzymology / Q000469:parasitology; D006801:Humans; D008958:Models, Molecular; D010654:Phenylbutyrates / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D010963:Plasmodium falciparum / Q000187:drug effects / Q000201:enzymology / Q000254:growth & development; D011480:Protease Inhibitors / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D015800:Protozoan Proteins; D013379:Substrate Specificity; D013816:Thermodynamics; D013844:Thiazoles / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology / Q000633:toxicity
chemical_list: D000577:Amides; D000962:Antimalarials; D002863:Chromogenic Compounds; C430091:KNI 764; C435823:KNI-727; C435824:KNI-840; D010654:Phenylbutyrates; D011480:Protease Inhibitors; D015800:Protozoan Proteins; D013844:Thiazoles; C034940:3-amino-2-hydroxy-4-phenylbutanoic acid; D016282:Aspartic Acid Endopeptidases; C100637:plasmepsin II