ABSTRACT
Parasite resistance to antimalarial drugs is a serious threat to human health, and novel agents that act on enzymes essential for parasite metabolism, such as proteases, are attractive targets for drug development. Recent studies have shown that clinically utilized human immunodeficiency virus (HIV) protease inhibitors can inhibit the in vitro growth of Plasmodium falciparum at or below concentrations found in human plasma after oral drug administration. The most potent in vitro antimalarial effects have been obtained for parasites treated with saquinavir, ritonavir, or lopinavir, findings confirmed in this study for a genetically distinct P. falciparum line (3D7). To investigate the potential in vivo activity of antiretroviral protease inhibitors (ARPIs) against malaria, we examined the effect of ARPI combinations in a murine model of malaria. In mice infected with Plasmodium chabaudi AS and treated orally with ritonavir-saquinavir or ritonavir-lopinavir, a delay in patency and a significant attenuation of parasitemia were observed. Using modeling and ligand docking studies we examined putative ligand binding sites of ARPIs in aspartyl proteases of P. falciparum (plasmepsins II and IV) and P. chabaudi (plasmepsin) and found that these in silico analyses support the antimalarial activity hypothesized to be mediated through inhibition of these enzymes. In addition, in vitro enzyme assays demonstrated that P. falciparum plasmepsins II and IV are both inhibited by the ARPIs saquinavir, ritonavir, and lopinavir. The combined results suggest that ARPIs have useful antimalarial activity that may be especially relevant in geographical regions where HIV and P. falciparum infections are both endemic.
Subject(s)
HIV Protease Inhibitors/pharmacology , Malaria/drug therapy , Plasmodium falciparum/drug effects , Animals , Aspartic Acid Endopeptidases/chemistry , Binding Sites , Crystallization , Female , HIV Protease Inhibitors/blood , HIV Protease Inhibitors/therapeutic use , Hemoglobins/metabolism , Mice , Mice, Inbred C57BL , Protein Conformation , Protozoan ProteinsABSTRACT
The World Health Organization is sponsoring major treatment programs with the aim of controlling helminth infection throughout the tropical world. Prominent among the anthelmintics recommended for use in these programs are drugs in the benzimidazole (BZ) class. Resistance to these drugs has been associated with polymorphisms in the beta-tubulin gene. We have cloned and sequenced the beta-tubulin genes of Strongyloides stercoralis and Strongyloides ratti and have proceeded to develop a protocol for genotyping single worms for polymorphisms in beta-tubulin. Our findings indicate that S. ratti has a single beta-tubulin gene, making DNA sequence analysis of a single larva PCR product a feasible means of studying BZ resistance in these species. Our genotyping test allows the identification of polymorphisms at codons 167, 198, and 200 in the Strongyloides beta-tubulin gene, thus enabling survey for BZ resistant genotypes.
