Abstract
Cysteine protease inhibitors are being studied as possible new antimalarial agents. To evaluate the potential for resistance to these compounds, we subjected chloroquine-resistant (W2 strain) Plasmodium falciparum to increasing concentrations of a vinyl sulfone cysteine protease inhibitor. After incubation with 1-200 nm morpholine urea-leucine-homophenylalanine-phenyl vinyl sulfone over approximately 8 months, highly resistant parasites ( approximately 100-fold increases in IC(50)) were selected. The vinyl sulfone-resistant parasites were also resistant to related peptidyl inhibitors, but had only modest ( approximately 2-fold) decreases in sensitivity to other cysteine protease inhibitors. Compared with the parental strain, resistant parasites showed no changes in multiplication rates, but elevations in cysteine protease activity, falcipain-2 and falcipain-3 copy numbers, transcription of falcipain genes, and levels of these target proteases in trophozoites. Resistant parasites grown in the absence of the vinyl sulfone for 12 weeks showed partial reversion, with increased inhibitor sensitivity and apparent decreases in copy numbers of falcipain-2 and falcipain-3. The sequences of falcipain-1, falcipain-2, and falcipain-3 were identical in sensitive and resistant parasites. The accumulation of a vinyl sulfone inhibitor was decreased approximately 9-fold in resistant parasites. In summary, parasites resistant to a cysteine protease inhibitor were selected, although the acquisition of high level resistance required extended exposure to the inhibitor and this resistance was somewhat unstable. Resistance was specific for the type of protease inhibitor used for the selection and appeared to be mediated both by alterations in inhibitor transport and by a previously unidentified mechanism in P. falciparum, the amplification of genes encoding targets of enzyme inhibitors.
Highlights
Malaria continues to be a challenging health problem, infecting hundreds of millions of people and resulting in 1-3 million deaths annually [1]
For the selection of vinyl sulfone-resistant mutants, we evaluated the W2 strain of P
The multiplication rates of three vinyl sulfone resistant clones were compared with the parental isolate
Summary
Malaria continues to be a challenging health problem, infecting hundreds of millions of people and resulting in 1-3 million deaths annually [1]. There is a great need to develop new antimalarial agents. Among potential new targets for antimalarial chemotherapy are proteases that hydrolyze hemoglobin in erythrocytic parasites [2, 3]. Two P. falciparum cysteine proteases, falcipain-2 [8] and falcipain-3 [9] have been characterized as hemoglobinases. They are of particular interest as therapeutic targets, as falcipain inhibitors, including peptidyl fluoromethyl ketones and vinyl sulfones, blocked the hydrolysis of hemoglobin and development by cultured erythrocytic parasites [11, 12] and cured mice infected with otherwise lethal malaria infections [13, 14]. The disruption of the falcipain-2 gene led to diminished hemoglobin hydrolysis in trophozoites, confirming the role of this enzyme as a hemoglobinase[15]
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call