Identification of antimalarial leads with dual falcipain-2 and falcipain-3 inhibitory activity

Falcipains (FPs), cysteine proteases in the malarial parasite, are emerging as the promising antimalarial drug targets. In order to identify novel FP inhibitors, we generated a pharmacophore derived from the reported co-crystal structures of inhibitors of Plasmodium falciparum Falcipain-3 to screen the ZINC library. Further, the filters were applied for dock score, drug-like characters, and clustering of similar structures. Sixteen molecules were purchased and subject to in vitro enzyme (FP-2 and FP-3) inhibition assays. Two compounds showed in vitro inhibition of FP-2 and FP-3 at low µM concentration. The selectivity of the inhibitors can be explained based on the predicted interactions of the molecule in the active site. Further, the inhibitors were evaluated in a functional assay and were found to induce morphological changes in line with their mode of action arresting Plasmodium development. Compound 15 was most potent inhibitor identified in this study.     

Biochemical Screening of Five Protein Kinases from Plasmodium falciparum against 14,000 Cell-Active Compounds

In 2010 the identities of thousands of anti-Plasmodium compounds were released publicly to facilitate malaria drug development. Understanding these compounds’ mechanisms of action—i.e., the specific molecular targets by which they kill the parasite—would further facilitate the drug development process. Given that kinases are promising anti-malaria targets, we screened ~14,000 cell-active compounds for activity against five different protein kinases. Collections of cell-active compounds from GlaxoSmithKline (the ~13,000-compound Tres Cantos Antimalarial Set, or TCAMS), St. Jude Children’s Research Hospital (260 compounds), and the Medicines for Malaria Venture (the 400-compound Malaria Box) were screened in biochemical assays of Plasmodium falciparum calcium-dependent protein kinases 1 and 4 (CDPK1 and CDPK4), mitogen-associated protein kinase 2 (MAPK2/MAP2), protein kinase 6 (PK6), and protein kinase 7 (PK7). Novel potent inhibitors (IC₅₀ < 1 μM) were discovered for three of the kinases: CDPK1, CDPK4, and PK6. The PK6 inhibitors are the most potent yet discovered for this enzyme and deserve further scrutiny. Additionally, kinome-wide competition assays revealed a compound that inhibits CDPK4 with few effects on ~150 human kinases, and several related compounds that inhibit CDPK1 and CDPK4 yet have limited cytotoxicity to human (HepG2) cells. Our data suggest that inhibiting multiple Plasmodium kinase targets without harming human cells is challenging but feasible.     

Antimalarial activity of thiosemicarbazones and purine derived nitriles

Malaria is a devastating illness caused by multiple species of the Plasmodium genus. The parasite’s falcipain proteases have been extensively studied as potential drug targets. Here we report the testing of two established cysteine protease inhibitor scaffolds against both chloroquine sensitive and chloroquine resistant parasites. A subset of purine derived nitriles killed the parasite with moderate potency, and these inhibitors do not seem to exert their antiproliferative effects as cysteine protease inhibitors. Compound potency was determined to be similar against both parasite strains, indicating a low probability of cross resistance with chloroquine. These compounds represent a novel antimalarial scaffold, and a potential starting point for the development new inhibitors.     

Semisynthesis,cytotoxicity, antimalarial evaluation and structure-activity relationship of two series of triterpene derivatives

In this report, we describe the semisynthesis of two series of ursolic and betulinic acid derivatives through designed by modifications at the C-3 and C-28 positions and demonstrate their antimalarial activity against chloroquine-resistant P. falciparum (W2 strain). Structural modifications at C-3 were more advantageous to antimalarial activity than simultaneous modifications at C-3 and C-28 positions. The ester derivative, 3β-butanoyl betulinic acid (7b), was the most active compound (IC₅₀?=?3.4?µM) and it did not exhibit cytotoxicity against VERO nor HepG2 cells (CC₅₀?>?400?µM), showing selectivity towards parasites (selectivity index?>?117.47). In combination with artemisinin, compound 7b showed an additive effect (CI?=?1.14). While docking analysis showed a possible interaction of 7b with the Plasmodium protease PfSUB1, with an optimum binding affinity of ?7.02?kcal/mol, the rather low inhibition displayed on a Bacillus licheniformis subtilisin A protease activity assay (IC₅₀?=?93?µM) and the observed accumulation of ring forms together with a delay of appearance of trophozoites in vitro suggests that the main target of 3β-butanoyl betulinic acid on Plasmodium may be related to other molecules and processes pertaining to the ring stage. Therefore, compound 7b is the most promising compound for further studies on antimalarial chemotherapy. The results obtained in this study provide suitable information about scaffolds to develop novel antimalarials from natural sources.     

Inhibition of Plasmodium falciparum cysteine proteases by the sugarcane cystatin CaneCPI-4

Malaria is a disease caused by Plasmodium parasites that affects hundreds of millions of people. Plasmodium proteases are involved in invasion, erythrocyte egress and degradation of host proteins. Falcipains are well-studied cysteine peptidases located in P. falciparum food vacuoles that participate in hemoglobin degradation. Cystatins are natural cysteine protease inhibitors that are implicated in a wide range of regulatory processes. Here, we report that a cystatin from sugarcane, CaneCPI-4, is selectively internalized into P. falciparum infected erythrocytes and is not processed by the parasite proteolytic machinery. Furthermore, we demonstrated the inhibition of P. falciparum cysteine proteases by CaneCPI-4, suggesting that it can exert inhibitory functions inside the parasites. The inhibition of the proteolytic activity of parasite cells is specific to this cystatin, as the addition of an anti-CaneCPI-4 antibody completely abolished the inhibition. We extended the studies to recombinant falcipain-2 and falcipain-3 and demonstrated that CaneCPI-4 strongly inhibits these enzymes, with IC₅₀ values of 12 nM and 42 nM, respectively. We also demonstrated that CaneCPI-4 decreased the hemozoin formation in the parasites, affecting the parasitemia. Taken together, this study identified a natural molecule as a potential antimalarial that specifically targets falcipains and also contributes to a better understanding of macromolecule acquisition by Plasmodium falciparum infected RBCs.     

Treatment of Erythrocytes with the 2-Cys Peroxiredoxin Inhibitor,Conoidin A,Prevents the Growth of Plasmodium falciparum and Enhances Parasite Sensitivity to Chloroquine

The human erythrocyte contains an abundance of the thiol-dependant peroxidase Peroxiredoxin-2 (Prx2), which protects the cell from the pro-oxidant environment it encounters during its 120 days of life in the blood stream. In malarial infections, the Plasmodium parasite invades red cells and imports Prx2 during intraerythrocytic development, presumably to supplement in its own degradation of peroxides generated during cell metabolism, especially hemoglobin (Hb) digestion. Here we demonstrate that an irreversible Prx2 inhibitor, Conoidin A (2,3-bis(bromomethyl)-1,4-dioxide-quinoxaline; BBMQ), has potent cytocidal activity against cultured P. falciparum. Parasite growth was also inhibited in red cells that were treated with BBMQ and then washed prior to parasite infection. These cells remained susceptible to merozoite invasion, but failed to support normal intraerythrocytic development. In addition the potency of chloroquine (CQ), an antimalarial drug that prevents the detoxification of Hb-derived heme, was significantly enhanced in the presence of BBMQ. CQ IC₅₀ values decreased an order of magnitude when parasites were either co-incubated with BBMQ, or introduced into BBMQ-pretreated cells; these effects were equivalent for both drug-resistant and drug-sensitive parasite lines. Together these results indicate that treatment of red cells with BBMQ renders them incapable of supporting parasite growth and increases parasite sensitivity to CQ. We also propose that molecules such as BBMQ that target host cell proteins may constitute a novel host-directed therapeutic approach for treating malaria.     

Small Molecule Screen for Candidate Antimalarials Targeting Plasmodium Kinesin-5

Plasmodium falciparum and vivax are responsible for the majority of malaria infections worldwide, resulting in over a million deaths annually. Malaria parasites now show measured resistance to all currently utilized drugs. Novel antimalarial drugs are urgently needed. The Plasmodium Kinesin-5 mechanoenzyme is a suitable “next generation” target. Discovered via small molecule screen experiments, the human Kinesin-5 has multiple allosteric sites that are “druggable.” One site in particular, unique in its sequence divergence across all homologs in the superfamily and even within the same family, exhibits exquisite drug specificity. We propose that Plasmodium Kinesin-5 shares this allosteric site and likewise can be targeted to uncover inhibitors with high specificity. To test this idea, we performed a screen for inhibitors selective for Plasmodium Kinesin-5 ATPase activity in parallel with human Kinesin-5. Our screen of nearly 2000 compounds successfully identified compounds that selectively inhibit both P. vivax and falciparum Kinesin-5 motor domains but, as anticipated, do not impact human Kinesin-5 activity. Of note is a candidate drug that did not biochemically compete with the ATP substrate for the conserved active site or disrupt the microtubule-binding site. Together, our experiments identified MMV666693 as a selective allosteric inhibitor of Plasmodium Kinesin-5; this is the first identified protein target for the Medicines of Malaria Venture validated collection of parasite proliferation inhibitors. This work demonstrates that chemical screens against human kinesins are adaptable to homologs in disease organisms and, as such, extendable to strategies to combat infectious disease.     

Inhibition of Plasmepsin V Activity Demonstrates Its Essential Role in Protein Export,PfEMP1 Display,and Survival of Malaria Parasites

The malaria parasite Plasmodium falciparum exports several hundred proteins into the infected erythrocyte that are involved in cellular remodeling and severe virulence. The export mechanism involves the Plasmodium export element (PEXEL), which is a cleavage site for the parasite protease, Plasmepsin V (PMV). The PMV gene is refractory to deletion, suggesting it is essential, but definitive proof is lacking. Here, we generated a PEXEL-mimetic inhibitor that potently blocks the activity of PMV isolated from P. falciparum and Plasmodium vivax. Assessment of PMV activity in P. falciparum revealed PEXEL cleavage occurs cotranslationaly, similar to signal peptidase. Treatment of P. falciparum–infected erythrocytes with the inhibitor caused dose-dependent inhibition of PEXEL processing as well as protein export, including impaired display of the major virulence adhesin, PfEMP1, on the erythrocyte surface, and cytoadherence. The inhibitor killed parasites at the trophozoite stage and knockdown of PMV enhanced sensitivity to the inhibitor, while overexpression of PMV increased resistance. This provides the first direct evidence that PMV activity is essential for protein export in Plasmodium spp. and for parasite survival in human erythrocytes and validates PMV as an antimalarial drug target.     

Structural characterization of the Plasmodium falciparum lactate transporter PfFNT alone and in complex with antimalarial compound MMV007839 reveals its inhibition mechanism

Plasmodium falciparum, the deadliest causal agent of malaria, caused more than half of the 229 million malaria cases worldwide in 2019. The emergence and spreading of frontline drug-resistant Plasmodium strains are challenging to overcome in the battle against malaria and raise urgent demands for novel antimalarial agents. The P. falciparum formate–nitrite transporter (PfFNT) is a potential drug target due to its housekeeping role in lactate efflux during the intraerythrocytic stage. Targeting PfFNT, MMV007839 was identified as a lead compound that kills parasites at submicromolar concentrations. Here, we present 2 cryogenic-electron microscopy (cryo-EM) structures of PfFNT, one with the protein in its apo form and one with it in complex with MMV007839, both at 2.3 Å resolution. Benefiting from the high-resolution structures, our study provides the molecular basis for both the lactate transport of PfFNT and the inhibition mechanism of MMV007839, which facilitates further antimalarial drug design.

The malaria parasite’s lactate transporter, PfFNT, is a potential drug target due to its housekeeping role in lactate efflux during the red blood cell stage. Cryo-EM structures of PfFNT alone or in complex with the anti-malarial drug MMV007839 reveal the mechanism of lactate transport and drug inhibition.     

In Silico Screening on the Three-dimensional Model of the Plasmodium vivax SUB1 Protease Leads to the Validation of a Novel Anti-parasite Compound

Widespread drug resistance calls for the urgent development of new antimalarials that target novel steps in the life cycle of Plasmodium falciparum and Plasmodium vivax. The essential subtilisin-like serine protease SUB1 of Plasmodium merozoites plays a dual role in egress from and invasion into host erythrocytes. It belongs to a new generation of attractive drug targets against which specific potent inhibitors are actively searched. We characterize here the P. vivax SUB1 enzyme and show that it displays a typical auto-processing pattern and apical localization in P. vivax merozoites. To search for small PvSUB1 inhibitors, we took advantage of the similarity of SUB1 with bacterial subtilisins and generated P. vivax SUB1 three-dimensional models. The structure-based virtual screening of a large commercial chemical compounds library identified 306 virtual best hits, of which 37 were experimentally confirmed inhibitors and 5 had K_i values of <50 μm for PvSUB1. Interestingly, they belong to different chemical families. The most promising competitive inhibitor of PvSUB1 (compound 2) was equally active on PfSUB1 and displayed anti-P. falciparum and Plasmodium berghei activity in vitro and in vivo, respectively. Compound 2 inhibited the endogenous PfSUB1 as illustrated by the inhibited maturation of its natural substrate PfSERA5 and inhibited parasite egress and subsequent erythrocyte invasion. These data indicate that the strategy of in silico screening of three-dimensional models to select for virtual inhibitors combined with stringent biological validation successfully identified several inhibitors of the PvSUB1 enzyme. The most promising hit proved to be a potent cross-inhibitor of PlasmodiumSUB1, laying the groundwork for the development of a globally active small compound antimalarial.     

Structural studies of vacuolar plasmepsins

Plasmepsins (PMs) are pepsin-like aspartic proteases present in different species of parasite Plasmodium. Four Plasmodium spp. (P. vivax, P. ovale, P. malariae, and the most lethal P. falciparum) are mainly responsible for causing human malaria that affects millions worldwide. Due to the complexity and rate of parasite mutation coupled with regional variations, and the emergence of P. falciparum strains which are resistant to antimalarial agents such as chloroquine and sulfadoxine/pyrimethamine, there is constant pressure to find new and lasting chemotherapeutic drug therapies. Since many proteases represent therapeutic targets and PMs have been shown to play an important role in the survival of parasite, these enzymes have recently been identified as promising targets for the development of novel antimalarial drugs. The genome of P. falciparum encodes 10 PMs (PMI, PMII, PMIV-X and histo-aspartic protease (HAP)), 4 of which (PMI, PMII, PMIV and HAP) reside within the food vacuole, are directly involved in degradation of human hemoglobin, and share 50-79% amino acid sequence identity. This review focuses on structural studies of only these four enzymes, including their orthologs in other Plasmodium spp.. Almost all original crystallographic studies were performed with PMII, but more recent work on PMIV, PMI, and HAP resulted in a more complete picture of the structure-function relationship of vacuolar PMs. Many structures of inhibitor complexes of vacuolar plasmepsins, as well as their zymogens, have been reported in the last 15 years. Information gained by such studies will be helpful for the development of better inhibitors that could become a new class of potent antimalarial drugs. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Sensitivity of Plasmodium falciparum to Antimalarial Drugs in Hainan Island,China

Pyronaridine and artesunate have been shown to be effective in falciparum malaria treatment. However, pyronaridine is rarely used in Hainan Island clinically, and artesunate is not widely used as a therapeutic agent. Instead, conventional antimalarial drugs, chloroquine and piperaquine, are used, explaining the emergence of chloroquine-resistant Plasmodium falciparum. In this article, we investigated the sensitivity of P. falciparum to antimalarial drugs used in Hainan Island for rational drug therapy. We performed in vivo (28 days) and in vitro tests to determine the sensitivity of P. falciparum to antimalarial drugs. Total 46 patients with falciparum malaria were treated with dihydroartemisinin/piperaquine phosphate (DUO-COTECXIN) and followed up for 28 day. The cure rate was 97.8%. The mean fever clearance time (22.5±10.6 hr) and the mean parasite clearance time (27.3±12.2 hr) showed no statistical significance with different genders, ages, temperatures, or parasite density (P>0.05). The resistance rates of chloroquine, piperaquine, pyronarididine, and artesunate detected in vitro were 71.9%, 40.6%, 12.5%, and 0%, respectively (P<0.0001). The resistance intensities decreased as follows: chloroquine>piperaquine>pyronarididine>artesunate. The inhibitory dose 50 (IC₅₀) was 3.77×10^-6 mol/L, 2.09×10^-6 mol/L, 0.09×10^-6 mol/L, and 0.05×10^-6 mol/L, and the mean concentrations for complete inhibition (CIMC) of schizont formation were 5.60×10^-6 mol/L, 9.26×10^-6 mol/L, 0.55×10^-6 mol/L, and 0.07×10^-6 mol/L, respectively. Dihydroartemisinin showed a strong therapeutic effect against falciparum malaria with a low toxicity.     

Double drugging of prolyl-tRNA synthetase provides a new paradigm for anti-infective drug development

Toxoplasmosis is caused by Toxoplasma gondii and in immunocompromised patients it may lead to seizures, encephalitis or death. The conserved enzyme prolyl-tRNA synthetase (PRS) is a validated druggable target in Toxoplasma gondii but the traditional ‘single target–single drug’ approach has its caveats. Here, we describe two potent inhibitors namely halofuginone (HFG) and a novel ATP mimetic (L95) that bind to Toxoplasma gondii PRS simultaneously at different neighbouring sites to cover all three of the enzyme substrate subsites. HFG and L95 act as one triple-site inhibitor in tandem and form an unusual ternary complex wherein HFG occupies the 3’-end of tRNA and the L-proline (L-pro) binding sites while L95 occupies the ATP pocket. These inhibitors exhibit nanomolar IC₅₀ and EC₅₀ values independently, and when given together reveal an additive mode of action in parasite inhibition assays. This work validates a novel approach and lays a structural framework for further drug development based on simultaneous targeting of multiple pockets to inhibit druggable proteins.     

3,5-Bis(benzylidene)-4-piperidones and related N-acyl analogs: A novel cluster of antimalarials targeting the liver stage of Plasmodium falciparum

Drug resistance is a major challenge in antimalarial chemotherapy. In addition, a complete cure of malaria requires intervention at various stages in the development of the parasite within the host. There are only a few antimalarials that target the liver stage of the Plasmodium species which is an essential part of the life cycle of the malarial parasite. We report a series of antimalarial 3,5-bis(benzylidene)-4-piperidones and related N-acyl analogs 1–5, a number of which exhibit potent in vitro growth-inhibiting properties towards drug-sensitive D6 and drug-resistant C235 strains of Plasmodium falciparum as well as inhibiting the liver stage development of the malarial life cycle. The compounds 2b (IC₅₀: 165 ng/mL), 3b (IC₅₀: 186 ng/mL), 5c (IC₅₀: 159 ng/mL) and 5d (IC₅₀: 93.5 ng/mL) emerged as lead molecules that inhibit liver stage Plasmodium berghei and are significantly more potent than chloroquine (IC₅₀: >2000 ng/mL) and mefloquine (IC₅₀: >2000 ng/mL) in this screen. All the compounds that showed potent inhibitory activity against the P. berghei liver stage were nontoxic to human HepG2 liver cells (IC₅₀: >2000 ng/mL). The compounds 5a and 5b exhibit comparable metabolic stability as chloroquine and mefloquine in human plasma and the most potent compound 5d demonstrated suitable permeability characteristics using the MDCK monolayer. These results emphasize the value of 3,5-bis(benzylidene)-4-piperidones as novel antimalarials for further drug development.     

Crystal Structure of the Plasmodium falciparum Thioredoxin Reductase–Thioredoxin Complex

Over the last decades, malaria parasites have been rapidly developing resistance against antimalarial drugs, which underlines the need for novel drug targets. Thioredoxin reductase (TrxR) is crucially involved in redox homeostasis and essential for Plasmodium falciparum. Here, we report the first crystal structure of P. falciparum TrxR bound to its substrate thioredoxin 1. Upon complex formation, the flexible C-terminal arm and an insertion loop of PfTrxR are rearranged, suggesting that the C-terminal arm changes its conformation during catalysis similar to human TrxR. Striking differences between P. falciparum and human TrxR are a Plasmodium-specific insertion and the conformation of the C-terminal arm, which lead to considerable differences in thioredoxin binding and disulfide reduction. Moreover, we functionally analyzed amino acid residues involved in substrate binding and in the architecture of the intersubunit cavity, which is a known binding site for disulfide reductase inhibitors. Cell biological experiments indicate that P. falciparum TrxR is indeed targeted in the parasite by specific inhibitors with antimalarial activity. Differences between P. falciparum and human TrxR and details on substrate reduction and inhibitor binding provide the first solid basis for structure-based drug development and lead optimization.     

Nima- and Aurora-related kinases of malaria parasites

Completion of the life cycle of malaria parasite requires a succession of developmental stages which vary greatly with respect to proliferation status, implying a tightly regulated control of the parasite's cell cycle, which remains to be understood at the molecular level. Progression of the eukaryotic cell cycle is controlled by members of mitotic kinase of the families CDK (cyclin-dependent kinases), Aurora, Polo and NIMA. Plasmodium parasites possess cyclin-dependent protein kinases and cyclins, which strongly suggests that some of the principles underlying cell cycle control in higher eukaryotes also operate in this organism. However, atypical features of Plasmodium cell cycle organization and important divergences in the composition of the cell cycle machinery suggest the existence of regulatory mechanisms that are at variance with those of higher eukaryotes. This review focuses on several recently described Plasmodium protein kinases related to the NIMA and Aurora kinase families and discusses their functional involvement in parasite's biology. Given their demonstrated essential roles in the erythrocytic asexual cycle and/or sexual stages, these enzymes represent novel potential drug targets for antimalarial intervention aiming at inhibiting parasite replication and/or blocking transmission of the disease. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).     

Antimalarial evaluation of copper(II) nanohybrid solids: inhibition of plasmepsin II, a hemoglobin-degrading malarial aspartic protease from Plasmodium falciparum

A new class of copper(II) nanohybrid solids, LCu(CH₃COO)₂ and LCuCl₂, have been synthesized and characterized by transmission electron microscopy, dynamic light scattering, and IR spectroscopy, and have been found to be capped by a bis(benzimidazole) diamide ligand (L). The particle sizes of these nanohybrid solids were found to be in the ranges 5–10 and 60–70 nm, respectively. These nanohybrid solids were evaluated for their in vitro antimalarial activity against a chloroquine-sensitive isolate of Plasmodium falciparum (MRC 2). The interactions between these nanohybrid solids and plasmepsin II (an aspartic protease and a plausible novel target for antimalarial drug development), which is believed to be essential for hemoglobin degradation by the parasite, have been assayed by UV–vis spectroscopy and inhibition kinetics using Lineweaver–Burk plots. Our results suggest that these two compounds have antimalarial activities, and the IC₅₀ values (0.025–0.032 μg/ml) are similar to the IC₅₀ value of the standard drug chloroquine used in the bioassay. Lineweaver–Burk plots for inhibition of plasmepsin II by LCu(CH₃COO)₂ and LCuCl₂ show that the inhibition is competitive with respect to the substrate. The inhibition constants of LCu(CH₃COO)₂ and LCuCl₂ were found to be 10 and 13 μM, respectively. The IC₅₀ values for inhibition of plasmepsin II by LCu(CH₃COO)₂ and LCuCl₂ were found to be 14 and 17 μM, respectively. Copper(II) metal capped by a benzimidazole group, which resembles the histidine group of copper proteins (galactose oxidase, β-hydroxylase), could provide a suitable anchoring site on the nanosurface and thus could be useful for inhibition of target enzymes via binding to the S1/S3 pocket of the enzyme hydrophobically. Both copper(II) nanohybrid solids were found to be nontoxic against human hepatocellular carcinoma cells and were highly selective for plasmepsin II versus human cathepsin D. The pivotal mechanism of antimalarial activity of these compounds via plasmepsin II inhibition in the P. falciparum malaria parasite is demonstrated.     

Malaria: Targeting parasite and host cell kinomes

Malaria still remains one of the deadliest infectious diseases, and has a tremendous morbidity and mortality impact in the developing world. The propensity of the parasites to develop drug resistance, and the relative reluctance of the pharmaceutical industry to invest massively in the developments of drugs that would offer only limited marketing prospects, are major issues in antimalarial drug discovery. Protein kinases (PKs) have become a major family of targets for drug discovery research in a number of disease contexts, which has generated considerable resources such as kinase-directed libraries and high throughput kinase inhibition assays. The phylogenetic distance between malaria parasites and their human host translates into important divergences in their respective kinomes, and most Plasmodium kinases display atypical properties (as compared to mammalian PKs) that can be exploited towards selective inhibition. Here, we discuss the taxon-specific kinases possessed by malaria parasites, and give an overview of target PKs that have been validated by reverse genetics, either in the human malaria parasite Plasmodium falciparum or in the rodent model Plasmodium berghei. We also briefly allude to the possibility of attacking Plasmodium through the inhibition of human PKs that are required for survival of this obligatory intracellular parasite, and which are targets for other human diseases.     

Plasmodium DDI1 is a potential therapeutic target and important chromatin-associated protein

DNA damage inducible 1 protein (DDI1) is involved in a variety of cellular processes including proteasomal degradation of specific proteins. All DDI1 proteins contain a ubiquitin-like (UBL) domain and a retroviral protease (RVP) domain. Some DDI1 proteins also contain a ubiquitin-associated (UBA) domain. The three domains confer distinct activities to DDI1 proteins. The presence of a RVP domain makes DDI1 a potential target of HIV protease inhibitors, which also block the development of malaria parasites. Hence, we investigated the DDI1 of malaria parasites to identify its roles during parasite development and potential as a therapeutic target. DDI1 proteins of Plasmodium and other apicomplexan parasites share the UBL-RVP domain architecture, and some also contain the UBA domain. Plasmodium DDI1 is expressed across all the major life cycle stages and is important for parasite survival, as conditional depletion of DDI1 protein in the mouse malaria parasite Plasmodium berghei and the human malaria parasite Plasmodium falciparum compromised parasite development. Infection of mice with DDI1 knock-down P. berghei was self-limiting and protected the recovered mice from subsequent infection with homologous as well as heterologous parasites, indicating the potential of DDI1 knock-down parasites as a whole organism vaccine. Plasmodium falciparum DDI1 (PfDDI1) is associated with chromatin and DNA-protein crosslinks. PfDDI1-depleted parasites accumulated DNA-protein crosslinks and showed enhanced susceptibility to DNA-damaging chemicals, indicating a role of PfDDI1 in removal of DNA-protein crosslinks. Knock-down of PfDDI1 increased susceptibility to the retroviral protease inhibitor lopinavir and antimalarial artemisinin, which suggests that simultaneous inhibition of DDI1 could potentiate antimalarial activity of these drugs. As DDI1 knock-down parasites confer protective immunity and it could be a target of HIV protease inhibitors, Plasmodium DDI1 is a potential therapeutic target for malaria control.     

Double-drug development against antioxidant enzymes from Plasmodium falciparum

Abstract

New drugs against malaria are urgently and continuously needed. Plasmodium parasites are exposed to higher fluxes of reactive oxygen species and need high activities of intracellular antioxidant systems. A most important antioxidative system consists of (di)thiols which are recycled by disulfide reductases (DR), namely both glutathione reductases (GR) of the malarial parasite Plasmodium falciparum and man, and the thioredoxin reductase (TrxR) of P. falciparum. The aim of our interdisciplinary research is to substantiate DR inhibitors as antimalarial agents. Such compounds are active per se but, in addition, they can reverse thiol-based resistance against other drugs in parasites. Reversal of drug resistance by DR inhibitors is currently investigated for the commonly used antimalarial drug chloroquine (CQ). Our recent strategy is based on the synthesis of inhibitors of the glutathione reductases from parasite and host erythrocyte. With the expectation of a synergistic or additive effect, double-headed prodrugs were designed to be directed against two different and essential functions of the malarial parasite P. falciparum, namely glutathione regeneration and heme detoxification. The prodrugs were prepared by linking bioreversibly a GR inhibitor to a 4-aminoquinoline moiety which is known to concentrate in the acidic food vacuole of parasites. Drug-enzyme interaction was correlated with antiparasitic action in vitro on strains resistant towards CQ and in vivo in Plasmodium berghei-infected mice as well as absence of cytotoxicity towards human cells. Because TrxR of P. falciparum was recently shown to be responsible for the residual glutathione disulfide-reducing capacity observed after GR inhibition in P. falciparum, future development of antimalarial drug-candidates that act by perturbing the redox equilibrium of parasites is based on the design of new double-drugs based on TrxR inhibitors as potential antimalarial drug candidates.