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.