Blood Stages Of Plasmodium Falciparum Research Articles

Understanding the significance of oxygen tension on the biology of Plasmodium falciparum blood stages: From the human body to the laboratory

Plasmodium falciparum undergoes sequestration within deep tissues of the human body, spanning multiple organ systems with differing oxygen (O2) concentrations. The parasite is exposed to an even greater range of O2 concentrations as it transitions from the human to the mosquito host, suggesting a high level of plasticity as it navigates these different environments. In this review, we explore factors that may contribute to the parasite’s response to different environmental O2 concentrations, recognizing that there are likely multiple pieces to this puzzle. We first review O2-sensing mechanisms, which exist in other apicomplexans such as Toxoplasma gondii and consider whether similar systems could exist in Plasmodium. Next, we review morphological and functional changes in P. falciparum’s mitochondrion during the asexual-to-sexual stage transition and discuss how these changes overlap with the parasite’s access to O2. We then delve into reactive oxygen species (ROS) as ROS production is influenced by O2 availability and oxidative stress impacts Plasmodium intraerythrocytic development. Lastly, given that the primary role of the red blood cell (RBC) is to deliver O2 throughout the body, we discuss how changes in the oxygenation status of hemoglobin, the RBC’s O2-carrying protein and key nutrient for Plasmodium, could also potentially impact the parasite’s growth during intraerythrocytic development. This review also highlights studies that have investigated P. falciparum biology under varying O2 concentrations and covers technical aspects related to P. falciparum cultivation in the lab, focusing on sources of technical variation that could alter the amount of dissolved O2 encountered by cells during in vitro experiments. Lastly, we discuss how culture systems can better replicate in vivo heterogeneity with respect to O2 gradients, propose ideas for further research in this area, and consider translational implications related to O2 and malaria.

High efficacy of chloroquine-derived bile salts in Pluronic F127 micelles against blood-stage Plasmodium falciparum

Colloidal nanocarriers can play a key role in the efficacious delivery of drugs, including antimalarials. Here, we investigated the ability of polymeric micelles of the block copolymer F127 to act as nanovehicles for two organic salts derived from chloroquine and human bile acids, namely, chloroquinium cholate (iCQP1) and chloroquinium glycocholate (iCQP1g). We have previously reported the strong in vitro antiplasmodial activity of these salts, which displayed IC50 values of 13 and 15 nM against blood forms of Plasmodium falciparum, respectively. By deriving from amphiphilic lipids, iCQP1 and iCQP1g also enclose the ability to act as surface-active ionic liquids (SAILs). The micellization properties of neat F127 and of the F127/SAIL mixtures were initially investigated to gain physicochemical insight into the interaction between polymer and bioactive SAILs, resorting to differential scanning calorimetry, surface tension measurements and dynamic light scattering. Micelle formation by F127 is an endothermic process strongly temperature and concentration dependent. Interestingly, this process is significantly changed when the molar fraction of SAIL (xSAIL) in the F127/SAIL mixture is varied between 0.33 and 0.90. Both SAILs favor the formation of mixed micelles by decreasing the micellization temperature, and (observed only when for xSAIL = 0.33) by synergistically decreasing the cmc. Concomitantly, the micellar size is reduced from 18 to 13 nm as xSAIL is increased from 0.33 to 0.90. Crucially, in vitro assays show that when the SAILs are loaded into F127 polymeric micelles, their antiplasmodial efficacy is substantially enhanced, with a significant drop in IC50, especially for the iCQP1/F127 system. This opens new possibilities for the nanoformulations of antimalarial compounds.

A picomolar inhibitor of the Plasmodium falciparum IPP pathway.

We identified MMV026468 as a picomolar inhibitor of blood-stage Plasmodium falciparum. Phenotyping assays, including isopentenyl diphosphate rescue of parasite growth inhibition, demonstrated that it targets MEP isoprenoid precursor biosynthesis. MMV026468-treated parasites showed an overall decrease in MEP pathway intermediates, which could result from inhibition of the first MEP enzyme DXS or steps prior to DXS such as regulation of the MEP pathway. Selection of MMV026468-resistant parasites lacking DXS mutations suggested that other targets are possible. The identification of MMV026468 could lead to a new class of antimalarial isoprenoid inhibitors.

Blood-stage malaria vaccine candidate RH5.1/Matrix-M in healthy Tanzanian adults and children; an open-label, non-randomised, first-in-human, single-centre, phase 1b trial

A blood-stage Plasmodium falciparum malaria vaccine would provide a second line of defence to complement partially effective or waning immunity conferred by the approved pre-erythrocytic vaccines. RH5.1 is a soluble protein vaccine candidate for blood-stage P falciparum, formulated with Matrix-M adjuvant to assess safety and immunogenicity in a malaria-endemic adult and paediatric population for the first time. We did a non-randomised, phase 1b, single-centre, dose-escalation, age de-escalation, first-in-human trial of RH5.1/Matrix-M in Bagamoyo, Tanzania. We recruited healthy adults (aged 18-45 years) and children (aged 5-17 months) to receive the RH5.1/Matrix-M vaccine candidate in the following three-dose regimens: 10 μg RH5.1 at 0, 1, and 2 months (Adults 10M), and the higher dose of 50 μg RH5.1 at 0 and 1 month and 10 μg RH5.1 at 6 months (delayed-fractional third dose regimen; Adults DFx). Children received either 10 μg RH5.1 at 0, 1, and 2 months (Children 10M) or 10 μg RH5.1 at 0, 1, and 6 months (delayed third dose regimen; Children 10D), and were recruited in parallel, followed by children who received the dose-escalation regimen (Children DFx) and children with higher malaria pre-exposure who also received the dose-escalation regimen (High Children DFx). All RH5.1 doses were formulated with 50 μg Matrix-M adjuvant. Primary outcomes for vaccine safety were solicited and unsolicited adverse events after each vaccination, along with any serious adverse events during the study period. The secondary outcome measures for immunogenicity were the concentration and avidity of anti-RH5.1 serum IgG antibodies and their percentage growth inhibition activity (GIA) in vitro, as well as cellular immunogenicity to RH5.1. All participants receiving at least one dose of vaccine were included in the primary analyses. This trial is registered at ClinicalTrials.gov, NCT04318002, and is now complete. Between Jan 25, 2021, and April 15, 2021, we recruited 12 adults (six [50%] in the Adults 10M group and six [50%] in the Adults DFx group) and 48 children (12 each in the Children 10M, Children 10D, Children DFx, and High Children DFx groups). 57 (95%) of 60 participants completed the vaccination series and 55 (92%) completed 22 months of follow-up following the third vaccination. Vaccinations were well-tolerated across both age groups. There were five serious adverse events involving four child participants during the trial, none of which were deemed related to vaccination. RH5-specific T cell and serum IgG antibody responses were induced by vaccination and purified total IgG showed in vitro GIA against P falciparum. We found similar functional quality (ie, GIA per μg RH5-specific IgG) across all age groups and dosing regimens at 14 days after the final vaccination; the concentration of RH5.1-specific polyclonal IgG required to give 50% GIA was 14·3 μg/mL (95% CI 13·4-15·2). 11 children were vaccinated with the delayed third dose regimen and showed the highest median anti-RH5 serum IgG concentration 14 days following the third vaccination (723 μg/mL [IQR 511-1000]), resulting in all 11 who received the full series showing greater than 60% GIA following dilution of total IgG to 2·5 mg/mL (median 88% [IQR 81-94]). The RH5.1/Matrix-M vaccine candidate shows an acceptable safety and reactogenicity profile in both adults and 5-17-month-old children residing in a malaria-endemic area, with all children in the delayed third dose regimen reaching a level of GIA previously associated with protective outcome against blood-stage P falciparum challenge in non-human primates. These data support onward efficacy assessment of this vaccine candidate against clinical malaria in young African children. The European and Developing Countries Clinical Trials Partnership; the UK Medical Research Council; the UK Department for International Development; the National Institute for Health and Care Research Oxford Biomedical Research Centre; the Division of Intramural Research, National Institute of Allergy and Infectious Diseases; the US Agency for International Development; and the Wellcome Trust.

Development of an improved blood-stage malaria vaccine targeting the essential RH5-CyRPA-RIPR invasion complex

Reticulocyte-binding protein homologue 5 (RH5), a leading blood-stage Plasmodium falciparum malaria vaccine target, interacts with cysteine-rich protective antigen (CyRPA) and RH5-interacting protein (RIPR) to form an essential heterotrimeric “RCR-complex”. We investigate whether RCR-complex vaccination can improve upon RH5 alone. Using monoclonal antibodies (mAbs) we show that parasite growth-inhibitory epitopes on each antigen are surface-exposed on the RCR-complex and that mAb pairs targeting different antigens can function additively or synergistically. However, immunisation of female rats with the RCR-complex fails to outperform RH5 alone due to immuno-dominance of RIPR coupled with inferior potency of anti-RIPR polyclonal IgG. We identify that all growth-inhibitory antibody epitopes of RIPR cluster within the C-terminal EGF-like domains and that a fusion of these domains to CyRPA, called “R78C”, combined with RH5, improves the level of in vitro parasite growth inhibition compared to RH5 alone. These preclinical data justify the advancement of the RH5.1 + R78C/Matrix-M™ vaccine candidate to Phase 1 clinical trial.

A pH Fingerprint Assay to Identify Inhibitors of Multiple Validated and Potential Antimalarial Drug Targets.

New drugs with novel modes of action are needed to safeguard malaria treatment. In recent years, millions of compounds have been tested for their ability to inhibit the growth of asexual blood-stage Plasmodium falciparum parasites, resulting in the identification of thousands of compounds with antiplasmodial activity. Determining the mechanisms of action of antiplasmodial compounds informs their further development, but remains challenging. A relatively high proportion of compounds identified as killing asexual blood-stage parasites show evidence of targeting the parasite's plasma membrane Na+-extruding, H+-importing pump, PfATP4. Inhibitors of PfATP4 give rise to characteristic changes in the parasite's internal [Na+] and pH. Here, we designed a "pH fingerprint" assay that robustly identifies PfATP4 inhibitors while simultaneously allowing the detection of (and discrimination between) inhibitors of the lactate:H+ transporter PfFNT, which is a validated antimalarial drug target, and the V-type H+ ATPase, which was suggested as a possible target of the clinical candidate ZY19489. In our pH fingerprint assays and subsequent secondary assays, ZY19489 did not show evidence for the inhibition of pH regulation by the V-type H+ ATPase, suggesting that it has a different mode of action in the parasite. The pH fingerprint assay also has the potential to identify protonophores, inhibitors of the acid-loading Cl- transporter(s) (for which the molecular identity(ies) remain elusive), and compounds that act through inhibition of either the glucose transporter PfHT or glycolysis. The pH fingerprint assay therefore provides an efficient starting point to match a proportion of antiplasmodial compounds with their mechanisms of action.

Tartrolon E, a secondary metabolite of a marine symbiotic bacterium, is a potent inhibitor of asexual and sexual Plasmodium falciparum.

Due to the spread of resistance to front-line artemisinin derivatives worldwide, there is a need for new antimalarials. Tartrolon E (TrtE), a secondary metabolite of a symbiotic bacterium of marine bivalve mollusks, is a promising antimalarial because it inhibits the growth of sexual and asexual blood stages of Plasmodium falciparum at sub-nanomolar levels. The potency of TrtE warrants further investigation into its mechanism of action, cytotoxicity, and ease with which parasites may evolve resistance to it.

In-vitro antimalarial activity of methanolic leaf- and stem-derived extracts from four Annonaceae plants

ObjectivePlants in the Annonaceae family are known for having abundant biologically active secondary metabolites. They have been used in alternative drugs for various diseases in several countries, for instance, the bark of Cananga odorata (Lam.) Hook and Thomson is used for Ophthalmic inflammation and wound healing in Malaysia. Extracts from the leaves and stems of four Annonaceae plants, namely Uvaria longipes (Craib) L.L.Zhou, Y.C.F.Su & R.M.K.Saunders, Dasymaschalon sp., Artabotrys burmanicus A.DC, and Marsypopetalum modestum (Pierre) B.Xue & R.M.K.Saunders were investigated for growth inhibitory activity against blood-stage Plasmodium falciparum growth in vitro and for non-specific cytotoxicity against normal peripheral blood mononuclear cells (PBMCs). Antimalarial activity was assessed by invasion inhibition assay and the percentage of infected red blood cells on blood smears were determined. Cytotoxicity was tested by culturing PBMCs with the extracts, and viabilities were determined by Annexin V/propidium iodide staining.ResultsA. burmanicus stem extract and M. modestum leaf extract were capable of inhibiting growth of P. falciparum when used at 200 µg/mL compared to chloroquine. The extracts at effective concentrations, did not affect the viability of PBMCs. These results support further need for characterization of active compounds from specific Annonaceae plants in order to exploit their components for potential malaria treatment.

The patatin-like phospholipase PfPNPLA2 is involved in the mitochondrial degradation of phosphatidylglycerol during Plasmodium falciparum blood stage development.

Plasmodium falciparum is an Apicomplexa responsible for human malaria, a major disease causing more than ½ million deaths every year, against which there is no fully efficient vaccine. The current rapid emergence of drug resistances emphasizes the need to identify novel drug targets. Increasing evidences show that lipid synthesis and trafficking are essential for parasite survival and pathogenesis, and that these pathways represent potential points of attack. Large amounts of phospholipids are needed for the generation of membrane compartments for newly divided parasites in the host cell. Parasite membrane homeostasis is achieved by an essential combination of parasite de novo lipid synthesis/recycling and massive host lipid scavenging. Latest data suggest that the mobilization and channeling of lipid resources is key for asexual parasite survival within the host red blood cell, but the molecular actors allowing lipid acquisition are poorly characterized. Enzymes remodeling lipids such as phospholipases are likely involved in these mechanisms. P. falciparum possesses an unusually large set of phospholipases, whose functions are largely unknown. Here we focused on the putative patatin-like phospholipase PfPNPLA2, for which we generated an glmS-inducible knockdown line and investigated its role during blood stages malaria. Disruption of the mitochondrial PfPNPLA2 in the asexual blood stages affected mitochondrial morphology and further induced a significant defect in parasite replication and survival, in particular under low host lipid availability. Lipidomic analyses revealed that PfPNPLA2 specifically degrades the parasite membrane lipid phosphatidylglycerol to generate lysobisphosphatidic acid. PfPNPLA2 knockdown further resulted in an increased host lipid scavenging accumulating in the form of storage lipids and free fatty acids. These results suggest that PfPNPLA2 is involved in the recycling of parasite phosphatidylglycerol to sustain optimal intraerythrocytic development when the host resources are scarce. This work strengthens our understanding of the complex lipid homeostasis pathways to acquire lipids and allow asexual parasite survival.

A conserved complex of microneme proteins mediates rhoptry discharge in Toxoplasma.

Apicomplexan parasites discharge specialized organelles called rhoptries upon host cell contact to mediate invasion. The events that drive rhoptry discharge are poorly understood, yet essential to sustain the apicomplexan parasitic life cycle. Rhoptry discharge appears to depend on proteins secreted from another set of organelles called micronemes, which vary in function from allowing host cell binding to facilitation of gliding motility. Here we examine the function of the microneme protein CLAMP, which we previously found to be necessary for Toxoplasma gondii host cell invasion, and demonstrate its essential role in rhoptry discharge. CLAMP forms a distinct complex with two other microneme proteins, the invasion-associated SPATR, and a previously uncharacterized protein we name CLAMP-linked invasion protein (CLIP). CLAMP deficiency does not impact parasite adhesion or microneme protein secretion; however, knockdown of any member of the CLAMP complex affects rhoptry discharge. Phylogenetic analysis suggests orthologs of the essential complex components, CLAMP and CLIP, are ubiquitous across apicomplexans. SPATR appears to act as an accessory factor in Toxoplasma, but despite incomplete conservation is also essential for invasion during Plasmodium falciparum blood stages. Together, our results reveal a new protein complex that mediates rhoptry discharge following host-cell contact.

STING activation promotes autologous type I interferon-dependent development of type 1 regulatory T cells during malaria.

The development of highly effective malaria vaccines and improvement of drug-treatment protocols to boost antiparasitic immunity are critical for malaria elimination. However, the rapid establishment of parasite-specific immune regulatory networks following exposure to malaria parasites hampers these efforts. Here, we identified stimulator of interferon genes (STING) as a critical mediator of type I interferon production by CD4+ T cells during blood-stage Plasmodium falciparum infection. The activation of STING in CD4+ T cells by cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) stimulated IFNB gene transcription, which promoted development of IL-10- and IFN-γ-coproducing CD4+ T (type I regulatory [Tr1]) cells. The critical role for type I IFN signaling for Tr1 cell development was confirmed in vivo using a preclinical malaria model. CD4+ T cell sensitivity to STING phosphorylation was increased in healthy volunteers following P. falciparum infection, particularly in Tr1 cells. These findings identified STING expressed by CD4+ T cells as an important mediator of type I IFN production and Tr1 cell development and activation during malaria.

A Pyridyl-Furan Series Developed from the Open Global Health Library Block Red Blood Cell Invasion and Protein Trafficking in Plasmodium falciparum through Potential Inhibition of the Parasite's PI4KIIIB Enzyme.

With the resistance increasing to current antimalarial medicines, there is an urgent need to discover new drug targets and to develop new medicines against these targets. We therefore screened the Open Global Health Library of Merck KGaA, Darmstadt, Germany, of 250 compounds against the asexual blood stage of the deadliest malarial parasite Plasmodium falciparum, from which eight inhibitors with low micromolar potency were found. Due to its combined potencies against parasite growth and inhibition of red blood cell invasion, the pyridyl-furan compound OGHL250 was prioritized for further optimization. The potency of the series lead compound (WEHI-518) was improved 250-fold to low nanomolar levels against parasite blood-stage growth. Parasites selected for resistance to a related compound, MMV396797, were also resistant to WEHI-518 as well as KDU731, an inhibitor of the phosphatidylinositol kinase PfPI4KIIIB, suggesting that this kinase is the target of the pyridyl-furan series. Inhibition of PfPI4KIIIB blocks multiple stages of the parasite's life cycle and other potent inhibitors are currently under preclinical development. MMV396797-resistant parasites possess an E1316D mutation in PfPKI4IIIB that clusters with known resistance mutations of other inhibitors of the kinase. Building upon earlier studies that showed that PfPI4KIIIB inhibitors block the development of the invasive merozoite parasite stage, we show that members of the pyridyl-furan series also block invasion and/or the conversion of merozoites into ring-stage intracellular parasites through inhibition of protein secretion and export into red blood cells.

Multifunctional IgG/IgM antibodies and cellular cytotoxicity are elicited by the full-length MSP1 SumayaVac-1 malaria vaccine

Radical control of malaria likely requires a vaccine that targets both the asymptomatic liver stages and the disease-causing blood stages of the human malaria parasite Plasmodium falciparum. While substantial progress has been made towards liver stage vaccines, the development of a blood stage vaccine is lagging behind. We have recently conducted a first-in-human clinical trial to evaluate the safety and immunogenicity of the recombinant, full-length merozoite surface protein 1 (MSP1FL) formulated with GLA-SE as adjuvant. Here, we show that the vaccine, termed SumayaVac-1, elicited both a humoral and cellular immune response as well as a recall T cell memory. The induced IgG and IgM antibodies were able to stimulate various Fc-mediated effector mechanisms associated with protection against malaria, including phagocytosis, release of reactive oxygen species, production of IFN-γ as well as complement activation and fixation. The multifunctional activity of the humoral immune response remained for at least 6 months after vaccination and was comparable to that of naturally acquired anti-MSP1 antibodies from semi-immune adults from Kenya. We further present evidence of SumayaVac-1 eliciting a recallable cellular cytotoxicity by IFN-γ producing CD8+ T cells. Our study revitalizes MSP1FL as a relevant blood stage vaccine candidate and warrants further evaluation of SumayaVac-1 in a phase II efficacy trial.

Global analysis of putative phospholipases in Plasmodium falciparum reveals an essential role of the phosphoinositide-specific phospholipase C in parasite maturation.

For its replication within red blood cells, the malaria parasite depends on a highly active and regulated lipid metabolism. Enzymes involved in lipid metabolic processes such as phospholipases are, therefore, potential drug targets. Here, using reverse genetics approaches, we show that only 1 out of the 19 putative phospholipases expressed in asexual blood stages of Plasmodium falciparum is essential for proliferation in vitro, pointing toward a high level of redundancy among members of this enzyme family. Using conditional mislocalization and gene disruption techniques, we show that this essential phosphoinositide-specific phospholipase C (PI-PLC, PF3D7_1013500) has a previously unrecognized essential role during intracellular parasite maturation, long before its previously perceived role in parasite egress and invasion. Subsequent lipidomic analysis suggests that PI-PLC mediates cleavage of phosphatidylinositol bisphosphate (PIP2) in schizont-stage parasites, underlining its critical role in regulating phosphoinositide levels in the parasite. IMPORTANCE The clinical symptoms of malaria arise due to repeated rounds of replication of Plasmodium parasites within red blood cells (RBCs). Central to this is an intense period of membrane biogenesis. Generation of membranes not only requires de novo synthesis and acquisition but also the degradation of phospholipids, a function that is performed by phospholipases. In this study, we investigate the essentiality of the 19 putative phospholipase enzymes that the human malaria parasite Plasmodium falciparum expresses during its replication within RBCs. We not only show that a high level of functional redundancy exists among these enzymes but, at the same time, also identify an essential role for the phosphoinositide-specific phospholipase C in parasite development and cleavage of the phospholipid phosphatidylinositol bisphosphate.

Activity of Ivermectin and Its Metabolites against Asexual Blood Stage Plasmodium falciparum and Its Interactions with Antimalarial Drugs.

Ivermectin is an endectocide used widely to treat a variety of internal and external parasites. Field trials of ivermectin mass drug administration for malaria transmission control have demonstrated a reduction of Anopheles mosquito survival and human malaria incidence. Ivermectin will mostly be deployed together with artemisinin-based combination therapies (ACT), the first-line treatment of falciparum malaria. It has not been well established if ivermectin has activity against asexual stage Plasmodium falciparum or if it interacts with the parasiticidal activity of other antimalarial drugs. This study evaluated antimalarial activity of ivermectin and its metabolites in artemisinin-sensitive and artemisinin-resistant P. falciparum isolates and assessed in vitro drug-drug interaction with artemisinins and its partner drugs. The concentration of ivermectin causing half of the maximum inhibitory activity (IC50) on parasite survival was 0.81 μM with no significant difference between artemisinin-sensitive and artemisinin-resistant isolates (P = 0.574). The ivermectin metabolites were 2-fold to 4-fold less active than the ivermectin parent compound (P < 0.001). Potential pharmacodynamic drug-drug interactions of ivermectin with artemisinins, ACT-partner drugs, and atovaquone were studied in vitro using mixture assays providing isobolograms and derived fractional inhibitory concentrations. There were no synergistic or antagonistic pharmacodynamic interactions when combining ivermectin and antimalarial drugs. In conclusion, ivermectin does not have clinically relevant activity against the asexual blood stages of P. falciparum. It also does not affect the in vitro antimalarial activity of artemisinins or ACT-partner drugs against asexual blood stages of P. falciparum.

Synthesis and In vitro Efficacy of Tetracyclic Benzothiazepines Against Blood-Stage Plasmodium falciparum and Liver-Stage P. berghei.

A series of novel, substituted tetracyclic benzothiazepines were designed and prepared in an effort to optimize the potency of this chemical class against drug-resistant strains of the malaria parasite. Tetracyclic benzothiazepines bearing structural modification at seven distinct positions within the structure were synthesized in Knoevenagel condensation reactions followed by sequential intermolecular thio-Michael and then intramolecular imine formation reactions. Following purification and chemical characterization, the novel compounds were tested for in vitro efficacy against blood-stage P. falciparum and liver-stage P. berghei and also for in vivo efficacy against P. berghei. Benzothiazepines bearing structural modification at the sulfur atom and at the three carbocycles within the molecule were successfully synthesized. The majority of analogs inhibited bloodstage P. falciparum with submicromolar IC50 values. The potency of an 8-methoxy-substituted analog 12 exceeded that of chloroquine in all three P. falciparum strains tested. The parent benzothiazepine 1 possessed liver-stage activity, inhibiting P. berghei sporozoites infecting HepG2 cells with an IC50 of 106.4 nM and an IC90 of 408.9 nM, but failed to enhance the longevity of P. berghei infected mice compared to the controls. Compounds displayed modest toxicity toward HepG2 cells and were tolerated by mice at the highest dose tested, 640 mg/kg/dose once daily for three days. The tetracyclic benzothiazepine described, which inhibits P. berghei infected hepatic cells with an IC50 of 106.4 nM, would appear to warrant further investigation. Optimization of ADME properties may be required since the most active analogs are probably excessively lipophilic.

Development of Potent and Highly Selective Epoxyketone-Based Plasmodium Proteasome Inhibitors.

Here, we present remarkable epoxyketone-based proteasome inhibitors with low nanomolar in vitro potency for blood-stage Plasmodium falciparum and low cytotoxicity for human cells. Our best compound has more than 2,000-fold greater selectivity for erythrocytic-stage P. falciparum over HepG2 and H460 cells, which is largely driven by the accommodation of the parasite proteasome for a D-amino acid in the P3 position and the preference for a difluorobenzyl group in the P1 position. We isolated the proteasome from P. falciparum cell extracts and determined that the best compound is 171-fold more potent at inhibiting the β5 subunit of P. falciparum proteasome when compared to the same subunit of the human constitutive proteasome. These compounds also significantly reduce parasitemia in a P. berghei mouse infection model and prolong survival of animals by an average of 6 days. The current epoxyketone inhibitors are ideal starting compounds for orally bioavailable anti-malarial drugs.

Hemisynthetic alkaloids derived from trilobine are antimalarials with sustained activity in multidrug-resistant Plasmodium falciparum

Malaria eradication requires the development of new drugs to combat drug-resistant parasites. We identified bisbenzylisoquinoline alkaloids isolated from Cocculus hirsutus that are active against Plasmodium falciparum blood stages. Synthesis of a library of 94 hemi-synthetic derivatives allowed to identify compound 84 that kills multi-drug resistant clinical isolates in the nanomolar range (median IC50 ranging from 35 to 88nM). Chemical optimization led to compound 125 with significantly improved preclinical properties. 125 delays the onset of parasitemia in Plasmodium berghei infected mice and inhibits P.falciparum transmission stages invitro (culture assays), and invivo using membrane feeding assay in the Anopheles stephensi vector. Compound 125 also impairs P.falciparum development in sporozoite-infected hepatocytes, in the low micromolar range. Finally, by chemical pull-down strategy, we characterized the parasite interactome with trilobine derivatives, identifying protein partners belonging to metabolic pathways that are not targeted by the actual antimalarial drugs or implicated in drug-resistance mechanisms.

Quantifying pH in Malaria Using pHluorin and Flow Cytometry.

Intracellular pH (pHi) plays a critical role in the regulation of numerous biological functions where specific pH ranges are required for optimal operation within cells. Slight pH changes can impact the regulation of diverse molecular processes, including enzymatic activities, ion channels, and transporters, which all play a role in cell functions. Methods for quantifying pHi continue to evolve and include various optical methods using fluorescent pH indicators. Here, we provide a protocol to measure pHi in the cytosol of Plasmodium falciparum blood stage parasites by means of flow cytometry and using pHluorin2, a pH-sensitive fluorescent protein that has been introduced into the genome of the parasite.

RH5.1-CyRPA-Ripr antigen combination vaccine shows little improvement over RH5.1 in a preclinical setting.

RH5 is the leading vaccine candidate for the Plasmodium falciparum blood stage and has shown impact on parasite growth in the blood in a human clinical trial. RH5 binds to Ripr and CyRPA at the apical end of the invasive merozoite form, and this complex, designated RCR, is essential for entry into human erythrocytes. RH5 has advanced to human clinical trials, and the impact on parasite growth in the blood was encouraging but modest. This study assessed the potential of a protein-in-adjuvant blood stage malaria vaccine based on a combination of RH5, Ripr and CyRPA to provide improved neutralizing activity against P. falciparum in vitro. Mice were immunized with the individual RCR antigens to down select the best performing adjuvant formulation and rats were immunized with the individual RCR antigens to select the correct antigen dose. A second cohort of rats were immunized with single, double and triple antigen combinations to assess immunogenicity and parasite neutralizing activity in growth inhibition assays. The DPX® platform was identified as the best performing formulation in potentiating P. falciparum inhibitory antibody responses to these antigens. The three antigens derived from RH5, Ripr and CyRPA proteins formulated with DPX induced highly inhibitory parasite neutralising antibodies. Notably, RH5 either as a single antigen or in combination with Ripr and/or CyRPA, induced inhibitory antibodies that outperformed CyRPA, Ripr. An RCR combination vaccine may not induce substantially improved protective immunity as compared with RH5 as a single immunogen in a clinical setting and leaves the development pathway open for other antigens to be combined with RH5 as a next generation malaria vaccine.