Role of Plasmodium falciparum Digestive Vacuole Plasmepsins in the Specificity and Antimalarial Mode of Action of Cysteine and Aspartic Protease Inhibitors

Abstract

Hemoglobin (Hb) degradation is essential for the growth of the intraerythrocytic stages of malarial parasites. This process, which occurs inside an acidic digestive vacuole (DV), is thought to involve the action of four aspartic proteases, termed plasmepsins (PMs). These enzymes have received considerable attention as potential antimalarial drug targets. Leveraging the availability of a set of PM-knockout lines generated in Plasmodium falciparum, we report here that a wide range of previously characterized or novel aspartic protease inhibitors exert their antimalarial activities independently of their effect on the DV PMs. We also assayed compounds previously shown to inhibit cysteine proteases residing in the DV. The most striking observation was a ninefold increase in the potency of the calpain inhibitor N-acetyl-leucinyl-leucinyl-norleucinal (ALLN) against parasites lacking all four DV PMs. Genetic ablation of PM III or PM IV also decreased the level of parasite resistance to the ␤-hematin binding antimalarial chloroquine. On the basis of the findings of drug susceptibility and isobologram assays, as well as the findings of studies of the inhibition of Hb degradation, morphological analyses, and stage specificity, we conclude that the DV PMs and falcipain cysteine proteases act cooperatively in Hb hydrolysis. We also identify several aspartic protease inhibitors, designed to target DV PMs, which appear to act on alternative targets early in the intraerythrocytic life cycle. These include the potent diphenylurea compound GB-III-32, which was found to be fourfold less potent against a P. falciparum line overexpressing plasmepsin X than against the parental nontransformed parasite line. The identification of the mode of action of these inhibitors will be important for future antimalarial drug discovery efforts focusing on aspartic proteases.Plasmodium falciparum malaria continues to exert a tremendous burden on communities in tropical and subtropical regions of the world. Efforts to control this disease have been stymied by the acquisition of resistance by this parasite to key antimalarials, including chloroquine (CQ) and pyrimethaminesulfadoxine (60). Hemoglobin (Hb) degradation plays an essential role in malarial parasite development within infected red blood cells. As such, the parasite proteases involved in Hb degradation appear to be attractive targets for the development of novel antimalarials that are unaffected by the existing mechanisms of drug resistance.Hb digestion takes place in an acidic compartment, referred to as the digestive vacuole (DV; also known as the food vacuole) (23). This degradative process provides a source of amino acids and is thought to help maintain intracellular osmolarity during rapid parasite growth (40). Biochemical studies have implicated the DV aspartic proteases, termed plasmepsin (PMs), and the cysteine proteases, termed falcipains, as key mediators of this degradative process (24,51,55

Erythrocyte Lysis and Xenopus laevis Oocyte Rupture by Recombinant Plasmodium falciparum Hemolysin III

“…The formation of swollen vacuoles in recPfHly III-transfected parasites may be a result of the following: (i) recPfHly III may be an influx channel and overexpression results in vacuolar swelling; (ii) in addition to channel activity, recPfHly III may have a regulatory effect on proteases and overexpressing recPfHly III may have inhibited these proteases. Prior reports have shown that protease inhibition can cause swollen vacuole phenotypes (35,36). It is quite possible that overexpression of recPfHly III results in the accumulation of metabolic products.…”

In vitro and In vivo Antimalarial Activity of Amphiphilic Naphthothiazolium Salts with Amine-Bearing Side Chains

“…27 Hemoglobin is hydrolyzed by a multiprotein complex containing aminopeptidases, aspartic proteases (plasmepsins), and cysteine proteases (falcipains). 28,29 This degradation of large amounts of hemoglobin is believed to be critical to provide a source of amino acids and to help maintain intracellular osmolarity during rapid parasite growth. 30 However, the breakdown of …”

Identification of plasmepsin inhibitors as selective anti-malarial agents using ligand based drug design

“…4 An analysis of the binding site region of PM II reveals that the binding site regions of PM I, IV, and HAP show 84%, 68% and 39% identity, respectively, while there is a lower degree of identity between the other six PMs. 8 PM V, IX and X have been shown to be produced in intraerythrocytic parasites 1,[10][11][12][13] but to date, function has been elucidated only for PMV.…”

“…500 million people each year. 1 The disease is caused by parasites from the genus Plasmodium and the Plasmepsins (PMs) are a family of aspartyl proteases (AP) found in each of the five species of Plasmodium that affect humans (P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi). This study focuses on the identification of PM inhibitors of the most lethal species, P. falciparum.…”

“…This study focuses on the identification of PM inhibitors of the most lethal species, P. falciparum. 1 Selectivity over the human equivalent is an important consideration when developing inhibitors of parasite enzymes. The PMs have a varied degree of sequence identity with the human aspartyl proteases, the most similar being the lysosomal enzyme Cathepsin D (CatD).…”