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.

A Simple Monochromatic Flow Cytometric Assay for Assessment of Intraerythrocytic Development of Plasmodium falciparum

Background: Gold standard microscopic examination of P. falciparum intraerythrocytic stage remains an important process for staging and enumerating parasitized erythrocytes in culture; however, microscopy is laborious and its accuracy is dependent upon the skill of the examiner. Methods: In this study, we used ViSafe Green (VSG), which is a nucleic acid-binding fluorescent dye, to assess in vitro development of P. falciparum using flow cytometry. Results: Fluorescence intensity of VSG was found to depend on the developmental stage of parasites. Specifically, multiple-nuclei-containing schizonts were observed in the VSGhigh population, and growing trophozoites and ring-shaped forms were observed in the VSGintermediate and VSGlow populations. The efficacy of our VSG-based assay was found to be comparable to the microscopic examination method, and it demonstrated an ability to detect as low as 0.001% of the parasitemia estimated by Giemsa staining. Moreover, when applying VSG for antimalarial drug test, we were able to observe the growth inhibitory effect of dihydroartemisinin, the front-line drug for malaria therapy. Conclusions: Taken together, the results of this study suggest the VSG-based flow cytometric assay to be a simple and reliable assay for assessing P. falciparum malaria development in vitro.

A Simple Monochromatic Flow Cytometric Assay for Assessment of Intraerythrocytic Development of Plasmodium falciparum

Background Gold standard microscopic examination of P. falciparum intraerythrocytic stage remains an important process for staging and enumerating parasitized erythrocytes in culture; however, microscopy is laborious, and its accuracy is dependent upon the skill of the examiner.Methods In this study, we used ViSafe Green (VSG), which is a nucleic acid-binding fluorescent dye, to assess in vitro development of P. falciparum using flow cytometry.Results Fluorescence intensity of VSG was found to depend on the developmental stage of parasites. Specifically, multiple-nuclei-containing schizonts were observed in the VSG high population, and growing trophozoites and ring-shaped forms were observed in the VSG intermediate and VSG low populations. The VSG-based assay was found to be comparable to the microscopic examination method, and capable of detecting as few as 0.001% of the parasitemia estimated by Giemsa staining. Moreover, when applying VSG for antimalarial drug test, we were able to observe the growth inhibitory effect of dihydroartemisinin, the front-line drug for malaria therapy.Conclusions Taken together, the results of this study suggest the VSG-based flow cytometric assay to be a simple and reliable assay for assessing P. falciparum malaria development in vitro .

Antimalarial Quinoline Drugs Inhibit β-Hematin and Increase Free Hemin Catalyzing Peroxidative Reactions and Inhibition of Cysteine Proteases

Malaria caused by Plasmodium affects millions people worldwide. Plasmodium consumes hemoglobin during its intraerythrocytic stage leaving toxic heme. Parasite detoxifies free heme through formation of hemozoin (β-hematin) pigment. Proteolysis of hemoglobin and formation of hemozoin are two main targets for antimalarial drugs. Quinoline antimarial drugs and analogs (β-carbolines or nitroindazoles) were studied as inhibitors of β-hematin formation. The most potent inhibitors were quinacrine, chloroquine, and amodiaquine followed by quinidine, mefloquine and quinine whereas 8-hydroxyquinoline and β-carbolines had no effect. Compounds that inhibited β-hematin increased free hemin that promoted peroxidative reactions as determined with TMB and ABTS substrates. Hemin-catalyzed peroxidative reactions were potentiated in presence of proteins (i.e. globin or BSA) while antioxidants and peroxidase inhibitors decreased peroxidation. Free hemin increased by chloroquine action promoted oxidative reactions resulting in inhibition of proteolysis by three cysteine proteases: papain, ficin and cathepsin B. Glutathione reversed inhibition of proteolysis. These results show that active quinolines inhibit hemozoin and increase free hemin which in presence of H2O2 that abounds in parasite digestive vacuole catalyzes peroxidative reactions and inhibition of cysteine proteases. This work suggests a link between the action of quinoline drugs with biochemical processes of peroxidation and inhibition of proteolysis.

Triazole substitution of a labile amide bond stabilizes pantothenamides and improves their antiplasmodial potency

The biosynthesis of CoA from pantothenate and the utilization of CoA in essential biochemical pathways represent promising antimalarial drug targets. Pantothenamides, amide-bearing pantothenate analogues, have potential as antimalarials, but a serum enzyme called pantetheinase degrades pantothenamides, rendering them inactivein vivo. In this study we characterize a series of 19 compounds that mimic pantothenamides with a stable triazole group instead of the labile amide. Two of these pantothenamides are active against the intraerythrocytic stage parasite with IC50values of ∼50 nM and three others have sub-micromolar IC50values. We show that the compounds target CoA biosynthesis and/or utilization. We investigated one of the compounds for its ability to interact with thePlasmodium falciparumpantothenate kinase, the first enzyme involved in the conversion of pantothenate to CoA and show that the compound inhibits the phosphorylation of [14C]pantothenate by theP. falciparumpantothenate kinase, but the inhibition does not correlate with antiplasmodial activity. Furthermore, the compounds are not toxic to human cells and, importantly, are not degraded by pantetheinase.

Maduramicin Rapidly Eliminates Malaria Parasites and Potentiates the Gametocytocidal Activity of the Pyrazoleamide PA21A050

New strategies targetingPlasmodium falciparumgametocytes, the sexual-stage parasites that are responsible for malaria transmission, are needed to eradicate this disease. Most commonly used antimalarials are ineffective againstP. falciparumgametocytes, allowing patients to continue to be infectious for over a week after asexual parasite clearance. A recent screen for gametocytocidal compounds demonstrated that the carboxylic polyether ionophore maduramicin is active at low nanomolar concentrations againstP. falciparumsexual stages. In this study, we showed that maduramicin has an EC50(effective concentration that inhibits the signal by 50%) of 14.8 nM against late-stage gametocytes and significantly blocksin vivotransmission in a mouse model of malaria transmission. In contrast to other reported gametocytocidal agents, maduramicin acts rapidlyin vitro, eliminating gametocytes and asexual schizonts in less than 12 h without affecting uninfected red blood cells (RBCs). Ring stage parasites are cleared by 24 h. Within an hour of drug treatment, 40% of the normally crescent-shaped gametocytes round up and become spherical. The number of round gametocytes increases to >60% by 2 h, even before a change in membrane potential as monitored by MitoProbe DiIC1 (5) is detectable. Maduramicin is not preferentially taken up by gametocyte-infected RBCs compared to uninfected RBCs, suggesting that gametocytes are more sensitive to alterations in cation concentration than RBCs. Moreover, the addition of 15.6 nM maduramicin enhanced the gametocytocidal activity of the pyrazoleamide PA21A050, which is a promising new antimalarial candidate associated with an increase in intracellular Na+concentration that is proposed to be due to inhibition of PfATP4, a putative Na+pump. These results underscore the importance of cation homeostasis in sexual as well as asexual intraerythrocytic-stageP. falciparumparasites and the potential of targeting this pathway for drug development.

Interaction of Normal and Sickle Hemoglobins for Sodium Dodecylsulphate and Hydrogen Peroxide at pH 5.0 and 7.2

Clinical manifestations of malaria primarily result from proliferation of the parasite within the hosts’ erythrocytes. The malaria parasite digests hemoglobin within its digestive vacuole through a sequential metabolic process involving multiple proteases. The activities of these proteases could lead to the production of ROS which could lead to the death of the parasites due to the destruction of their membrane. The action of SDS on hemoglobins can be likened to the way malarial proteases destabilizes host hemoglobin. Hence, the study was designed to determine the binding parameters of SDS and H2O2 for normal, sickle trait carrier and sickle hemoglobins at pH 5.0 and 7.2 using UV-VIS Titration Spectrophotometry. Hb-SDS interactions were significantly different at pH 5.0 but were not at pH 7.2. Also, Hb-H2O2 interactions were statistically different at pH 5.0 and 7.2. The interactions suggest that HbA and HbS are easily destabilized than HbAS and that HbAS has more affinity for H2O2. These suggest a production of more ferryl intermediates or hydroxyl radicals. All these interactions may hinder the development of the malaria parasite at the intraerythrocytic stage and could likely account for a significant proportion of the mechanism that favours the resistance to malaria by individuals with HbAS.

In VitroandIn VivoAntiplasmodial Activities of Risedronate and Its Interference with Protein Prenylation in Plasmodium falciparum

ABSTRACTThe increasing resistance of malarial parasites to almost all available drugs calls for the identification of new compounds and the detection of novel targets. Here, we establish the antimalarial activities of risedronate, one of the most potent bisphosphonates clinically used to treat bone resorption diseases, against blood stages ofPlasmodium falciparum(50% inhibitory concentration [IC50] of 20.3 ± 1.0 μM). We also suggest a mechanism of action for risedronate against the intraerythrocytic stage ofP. falciparumand show that protein prenylation seems to be modulated directly by this drug. Risedronate inhibits the transfer of the farnesyl pyrophosphate group to parasite proteins, an effect not observed for the transfer of geranylgeranyl pyrophosphate. Ourin vivoexperiments further demonstrate that risedronate leads to an 88.9% inhibition of the rodent parasitePlasmodium bergheiin mice on the seventh day of treatment; however, risedronate treatment did not result in a general increase of survival rates.

Colorimetric High-Throughput Screen for Detection of Heme Crystallization Inhibitors

ABSTRACT Malaria infects 500 million people annually, a number that is likely to rise as drug resistance to currently used antimalarials increases. During its intraerythrocytic stage, the causative parasite, Plasmodium falciparum, metabolizes hemoglobin and releases toxic heme, which is neutralized by a parasite-specific crystallization mechanism to form hemozoin. Evidence suggests that chloroquine, the most successful antimalarial agent in history, acts by disrupting the formation of hemozoin. Here we describe the development of a 384-well microtiter plate screen to detect small molecules that can also disrupt heme crystallization. This assay, which is based on a colorimetric assay developed by Ncokazi and Egan (K. K. Ncokazi and T. J. Egan, Anal. Biochem. 338:306-319, 2005), requires no parasites or parasite-derived reagents and no radioactive materials and is suitable for a high-throughput screening platform. The assay's reproducibility and large dynamic range are reflected by a Z factor of 0.74. A pilot screen of 16,000 small molecules belonging to diverse structural classes was conducted. The results of the target-based assay were compared with a whole-parasite viability assay of the same small molecules to identify small molecules active in both assays.