The Plasmodium falciparum cytoplasmic translation apparatus: a promising therapeutic target not yet exploited by clinically approved anti-malarials
© The Author(s) 2018
- Received: 5 October 2018
- Accepted: 6 December 2018
- Published: 12 December 2018
The continued spectre of resistance to existing anti-malarials necessitates the pursuit of novel targets and mechanisms of action for drug development. One class of promising targets consists of the 80S ribosome and its associated components comprising the parasite translational apparatus. Development of translation-targeting therapeutics requires a greater understanding of protein synthesis and its regulation in the malaria parasite. Research in this area has been limited by the lack of appropriate experimental methods, particularly a direct measure of parasite translation.
An in vitro method directly measuring translation in whole-cell extracts from the malaria parasite Plasmodium falciparum, the PfIVT assay, and a historically-utilized indirect measure of translation, S35-radiolabel incorporation, were compared utilizing a large panel of known translation inhibitors as well as anti-malarial drugs.
Here, an extensive pharmacologic assessment of the PfIVT assay is presented, using a wide range of known inhibitors demonstrating its utility for studying activity of both ribosomal and non-ribosomal elements directly involved in translation. Further, the superiority of this assay over a historically utilized indirect measure of translation, S35-radiolabel incorporation, is demonstrated. Additionally, the PfIVT assay is utilized to investigate a panel of clinically approved anti-malarial drugs, many with unknown or unclear mechanisms of action, and show that none inhibit translation, reaffirming Plasmodium translation to be a viable alternative drug target. Within this set, mefloquine is unambiguously found to lack translation inhibition activity, despite having been recently mischaracterized as a ribosomal inhibitor.
This work exploits a direct and reproducible assay for measuring P. falciparum translation, demonstrating its value in the continued study of protein synthesis in malaria and its inhibition as a drug target.
- 80S ribosome
- In vitro translation assay
- Antimalarial drugs
Despite ongoing efforts in its treatment and prevention, malaria remains a severe global health burden, with nearly half the world’s population at risk, and incidence of the disease actually increasing in the most recent years for which data are available . Though malaria-related mortality has continued to decrease, the rise in incidence is particularly concerning in light of reduced investment worldwide in combatting malaria, combined with climate change and geopolitical instability that may contribute to a resurgence of the disease . One compounding factor in the battle to eliminate malaria is the persistent emergence of drug resistance in the malaria parasite Plasmodium falciparum . As combination therapies are the main defense against resistance, an important focus in therapeutic development is the identification of compounds with unique targets and novel mechanisms of action that are unlikely to be precluded by existing resistance mutations. Medicines for Malaria Venture (MMV) has recently demonstrated the potential of efforts directed at novel targets; two drugs currently showing great promise in clinical trials, SJ733 and cipargamin, inhibit the P. falciparum cation ATPase PfATP4, constituting a new class of drug [2, 3].
One promising avenue for development of a novel target class is the inhibition of the P. falciparum ribosome, as well as other components of the translational machinery responsible for protein synthesis. Translation inhibitors have exhibited great clinical success as potent antibiotics, and in fact, several, including doxycycline and azithromycin, have found additional application as anti-malarials, as they target ribosomes within the malaria parasite’s mitochondria and apicoplast, leading to loss of function of these organelles [4–6]. Interestingly, the P. falciparum cytoplasmic ribosome appears to occupy an evolutionary middle ground between prokaryotic and eukaryotic, differentiating it sufficiently from human ribosomes to yield a useful therapeutic window . Indeed, M5717 (previously DDD107498), a potent and highly selective inhibitor of the 80S ribosome interacting protein P. falciparum eukaryotic elongation factor 2 (PfEF2), is currently in first-in-human study, validating the potential of the P. falciparum translational apparatus as an effective target for anti-malarial drugs of this class .
To facilitate the identification of translation inhibitors, a P. falciparum whole-cell extract-based in vitro translation assay (PfIVT) was developed, and the technique successfully applied to detect small molecule inhibitors in the MMV Malaria Box . More recently, it has been suggested that the widely used drug mefloquine may inhibit the 80S ribosome of P. falciparum . In addition, many currently approved anti-malarial compounds lack a definitive mechanism of action, raising the possibility that some of these clinical therapies act through inhibition of translation. The aim of this study was to clarify which compounds truly exhibit inhibitory activity against the P. falciparum 80S ribosome and the associated translational apparatus. To do so, a panel of anti-malarial drugs (both clinical and pre-clinical) was compared with well-characterized inhibitors of translation and other defined control compounds in the PfIVT assay, as well as in the S35-radioabel incorporation assay, a historically utilized indirect measure of translation. Importantly, this demonstrated that none of the current clinical therapeutics inhibit translation, including mefloquine. Regardless, testing of tool compounds shows that the PfIVT assay is capable of identifying not only translation inhibitors that directly interact with the ribosome, but also inhibitors of other non-ribosomal components of the translational machinery, demonstrating the broad utility of the assay for identifying novel malaria therapeutics that target P. falciparum translation.
In vivo growth and in vitro translation measurements were performed using the same drug dilutions. The anti-malarial drugs chloroquine, dihydroartemisinin, lumefantrine, monodesethyl amodiaquine, piperaquine, primaquine, and quinine were a generous gift from Dr. Phil Rosenthal of UCSF. SJ733 was generously provided by Dr. Kip Guy of St. Jude Children’s Research Hospital. All other compounds were purchased from the indicated vendors: DDD107498 (Apexbio #A8711-5), mefloquine hydrochloride (Sigma-Aldrich #M2319), emetine (Sigma-Aldrich #E2375), cycloheximide (Fisher #AC35742-0010), MMV008270 (Vitas-M Laboratory #STK591252), actinomycin D (Sigma-Aldrich #A1410), tubercidin (Sigma-Aldrich #T0642), thapsigargin (Sigma-Aldrich #SML1845), ionomycin (Sigma-Aldrich #407951), thiostrepton (Sigma-Aldrich #598226), bruceantin (Toronto Research Chemicals #B689310), verrucarin A (Sigma-Aldrich #V4877), anisomycin (Sigma-Aldrich #A5862), homoharringtonine (Sigma-Aldrich #SML1091), lactimidomycin (EMD Millipore #506291), nagilactone C (BOC Sciences #24338-53-2), suramin sodium salt (Sigma-Aldrich #S2671), puromycin (Thermo Fisher #A1113803), halofuginone (Sigma-Aldrich #32481).
Plasmodium falciparum strain and culturing
Plasmodium falciparum W2 (MRA-157) was obtained from MR4. Parasites were grown in human erythrocytes (2% haematocrit) in RPMIc (RPMI 1640 media supplemented with 0.25% Albumax II (GIBCO Life Technologies), 2 g/L sodium bicarbonate, 0.1 mM hypoxanthine, 25 mM HEPES (pH 7.4), and 50 μg/L gentamicin), at 37 °C, 5% O2, and 5% CO2. Cells were synchronized with 5% sorbitol treatment for two generations to achieve high synchronicity.
Growth inhibition assays
2μL of serial drug dilutions in 100% DMSO were dispensed in triplicate to 96-well plates utilizing the LabCyte ECHO acoustic liquid handler. 198 µL of P. falciparum W2 cultures were added. Growth was initiated with ring-stage parasites at 0.8% parasitaemia and 0.5% haematocrit. Plates were incubated at 37 °C, 5% O2, and 5% CO2 for 72 h. Growth was terminated by fixation with 1% formaldehyde, and parasitized cells were stained with 50 nM YOYO-1 (Invitrogen). Parasitaemia was determined by flow cytometry on the BD LSRII, analysed using FlowJo software version 10, and EC50 curves were plotted by GraphPad Prism. Two biological replicates were performed for each drug.
Generation and quality control of extracts for Plasmodium falciparum in vitro translation assay
For PfIVT harvests, 1 L of synchronized parasite culture in 2–4% haematocrit was grown in two 500 mL HYPERFlask M vessels (Corning), and media was changed every 8–12 h, with the final media change at 4–8 h prior to harvest. Parasites were harvested in the late trophozoite stage at 15–20% parasitaemia by centrifugation for 5 min at 1500×g at room temperature, followed by removal of media and addition of ice-cold 0.025–0.05% final saponin in Buffer A (20 mM HEPES pH 8.0, 2 mM Mg(OAc)2, 120 mM KOAc). Due to variations between and within lots, saponin stocks were prepared in large volumes, aliquoted, and stored at − 20 °C. Percentage utilized for each batch of aliquots was determined empirically through pairwise testing of concentrations (1 for each HYPERFlask) and assessed via resulting activity of PfIVT extracts. Saponin-lysed pellets were centrifuged at 4 °C and 10,000×g for 10 min and washed twice with ice-cold Buffer A. Supernatant was carefully removed, and washed pellets were resuspended in an equal volume of Buffer B2 [20 mM HEPES pH8.0, 100 mM KOAc, 0.75 mM Mg(OAC)2, 2 mM DTT, 20% glycerol, 1X EDTA-free protease inhibitor cocktail (Roche)], flash frozen, and stored in − 80 °C freezer until the sample was ready to homogenize.
Frozen pellets were thawed on ice and added to a 3-mL Luer lock syringe, which was then secured onto a pre-chilled cell homogenizer containing a 4μm-clearance ball bearing (Isobiotec, Germany) that was pre-washed with ice-cold Buffer B2. Homogenate was passed between two syringes 20 times on ice, either by hand or by use of a custom robot built to accommodate the cell homogenizer . Lysate was immediately centrifuged at 4 °C and 16,000×g for 10 min, and the supernatant (the resulting PfIVT extract) was transferred to a fresh tube, with a small (100 μL) aliquot set aside for activity testing. Extracts and test aliquots were flash-frozen and stored at − 80 °C. Test aliquots from multiple harvests were thawed on ice and tested in batches in the PfIVT assay (see below) across a small range of magnesium concentrations with a 2-h incubation time, using a firefly luciferase reporter. Extracts that surpass the activity threshold of 104 relative luciferase units (RLU) were then thawed on ice and combined to generate large volume pools. Extract pools were flash-frozen in 200 μL aliquots and stored at − 80 °C. Extract pools were tested across a range of magnesium concentrations via PfIVT assay to determine the optimum magnesium concentration. Once magnesium concentration has been determined, pools are then tested in the PfIVT assay in 15 min incubation time points up to 150 min to determine the kinetics of the extract pool, and thus the appropriate incubation time for the pool (~ 75–80% of maximum signal, within the linear range of the extract’s kinetic curve). Kinetics must be separately assessed for each reporter used (i.e. if a nanoluciferase reporter is used instead of firefly luciferase).
Magnesium concentration assays
Baseline magnesium levels of the PfIVT extracts were measured using a magnesium-dependent enzyme-based colorimetric assay kit (Sigma-Aldrich #MAK026). Two biological replicates of a dilution series of each extract were tested in duplicate with each of two separate kits, following the protocol provided with the kit. In brief, 10 μL of each PfIVT extract (neat, or diluted 1:4 or 1:10 with ddH2O) added to 10 μL ddH2O, along with a standard curve, was combined with 50 μL of master reaction mix (35 μL magnesium assay buffer, 10 μL developer, 5 μL magnesium enzyme mix), and incubated for 10 min with shaking at 37 °C. 450 nm absorbance was read immediately after the initial incubation, and every 5 min thereafter on a Tecan plate reader until the highest A450 approached (but did not exceed) 1.5× the initial reading. Values were fitted to, and interpolated from, the standard curve using Prism GraphPad.
Plasmodium falciparum in vitro translation assay
Plasmodium falciparum in vitro translation (PfIVT) reactions were carried out in skirted v-bottom 96-well PCR plates (BioRad) and sealed with adhesive aluminum foil plate seals (Beckman Coulter, Indianapolis, IN, USA). 200 nL of drug in 100% DMSO was dispensed in duplicate to appropriate wells of the plate utilizing a Labcyte ECHO acoustic liquid handler. 19.8 μL of PfIVT reaction mix (per 20 μL: 14 μL extract, 1 μg T7-transcribed firefly luciferase mRNA, 10 µM amino acid mixture, 20 mM HEPES/KOH pH 8.0, 75 mM KOAc, 2 mM DTT, 0.5 mM ATP, 0.1 mM GTP, 20 mM creatine phosphate, 0.2 μg/μL creatine kinase, and the appropriate amount of Mg(OAc)2 as determined for the particular pool of extract) was then dispensed to each well using Rainin E4 12-channel electronic pipettes (Rainin Instruments, Oakland, CA, USA). Reactions were incubated at 37 °C for the appropriate amount of time as determined for the particular pool of extract. After incubation, the reactions were placed on ice, then quenched through transfer to a 96-well LUMITRAC 200 flat-bottom white assay plate (Greiner Bio-One, Monroe, NC, USA) containing 2 μL of 50 μM cycloheximide (dispensed using the Labcyte ECHO), then immediately centrifuged to combine the PfIVT reaction with the cycloheximide for a final concentration of 5 µM cycloheximide. Reactions were assayed using the Promega GloMax-Multi + microplate reader with a three-second delay and three-second integration after addition of 200 μL luciferin reagent dispensed at a speed of 200 μL/second (firefly luciferin reagent: 20 mM Tricine, 2.67 mM MgSO4 × 7H2O, 0.1 mM EDTA, 33.3 mM DTT, 530 μM ATP, 270 μM Acetyl CoEnzyme A, 1 mM d-Luciferin, 265 μM Magnesium Carbonate Hydroxide, pH 8.15). Three biological replicates were performed in duplicate for each drug. IC50 curves were plotted by GraphPad Prism.
Rabbit reticulocyte in vitro translation assay
Rabbit reticulocyte in vitro translation assays were performed as described in Ahyong et al.  with the exception that the final [DMSO] for MMV008270 was 2.5% and all other final [DMSO] = 0.55% IC50 curves were plotted by GraphPad Prism.
S35 incorporation assay
Synchronized parasites were cultured in 2% haematocrit at 10–15% parasitaemia, and MACS purified at the late trophozoite stage to remove uninfected erythrocytes using standard protocols. In brief, at least two LD MACS Separation columns (Miltenyi Biotech) per 50 mL of culture were washed with 1.25 mL of pre-warmed RPMIc. Next, cultures were added to the columns 5 mL at a time and allowed to gravity filter at 37 °C. Finally, the columns were rinsed with 2.5 mL of pre-warmed RPMIc, removed from the magnetic stand, and eluted with 2 mL of pre-warmed RPMIc.
Drug treatment and S35 labelling
1 μL of drug in 100% DMSO was dispensed to each well of a 96-well round-bottom culture plate utilizing the Labcyte ECHO acoustic liquid handler. 2 × 107 MACS-purified parasites in 199 μL of RPMIc were then added to each well. Parasites were incubated with drug for 2 h at 37 °C, 5% CO2, 5% O2. Next, samples were transferred from 96-well plates to 1.5 mL screw-cap microfuge tubes. 35 μCi of EasyTag™ Express S35 Protein Labeling Mix (Perkin Elmer) diluted to 10 μL with RPMIc was added to each tube. Reactions were incubated at 37 °C with mild shaking for 2 h.
Washing and lysis
After incubation, cells were pelleted and 160 μL of supernatant was removed. Parasites were then washed with 200 μL of ice-cold PBS containing 50 μM cycloheximide four times. After the final wash, all supernatant was removed and samples were resuspended in 15 μL of 2X SDS buffer (100 mM Tris–Cl pH 6.8, 4% SDS, 20% glycerol, 0.1 M DTT). Samples were boiled at 98 °C for 5 min and stored at − 20 °C.
Samples were thawed at room temperature, boiled for 5 min at 98 °C, and spun at max speed in a tabletop microcentrifuge for 10 min. 10 μL of supernatant per sample was placed on a 0.45μm nitrocellulose membrane (HAWP02400 from Millipore). Each membrane was washed 4 times with 15 mL of TBS-T then placed in a 20 mL HDPE scintillation vial (Fisher Scientific) with 8 mL of Ecoscint A (National Diagnostics). S35 counts were measured for 1 min using a Beckmann coulter, LS 6500 Multi-purpose Scintillation Counter. Three biological replicates were performed for each drug.
Mefloquine solubility assay
PfIVT extracts were incubated with a dilution series of mefloquine or DMSO control for 90 min. All PfIVT conditions were the same as above, except without addition of cycloheximide to stop translation. Reactions were centrifuged at 16,100×g for 10 min at room temperature; resulting supernatant was then filtered and added to cultures for the P. falciparum growth inhibition assays as described above.
Extract optimization and quality control for the Plasmodium falciparum in vitro translation assay
Probing different stages of translation in a Plasmodium falciparum cellular extract system using tool compounds
The process of translation may be binned into three main stages: initiation, elongation, and termination [12, 13]. In eukaryotes, this process is carried out by the 80S ribosome, comprised of a small (40S) and large (60S) subunit [12, 13]. To further validate the PfIVT assay and investigate its capacity to interrogate the entirety of the normal activity of the 80S ribosome (and thus identify drugs inhibiting all steps of the process of translation), an extensive panel of previously characterized translational inhibitors was tested, both in the PfIVT assay, as well as in the historically utilized S35-radiolabelled amino acid incorporation assay. In contrast to the PfIVT assay, which directly measures activity of the 80S ribosome and the associated translational apparatus, S35 incorporation is an indirect measure of translation, and, as such, is affected by upstream and parallel pathways not directly involved in ribosomal activity. For instance, the S35 incorporation assay is impacted by changes in cell viability, stress or other responses that alter proteostasis, and even changes to cellular import mechanisms or integrity of the parasite membrane, as the assay requires exogenous addition of radiolabelled amino acid. Despite the resulting ambiguity and often misleading nature of results generated, S35 incorporation has remained a commonly used assay for studying parasite translation in the absence of a better alternative .
Mechanism of action and species specificity of translation inhibitors
Mechanism of action
Eukaryotic (Pf cytoplasmic ribosomes)
Eukaryotic (Pf cytoplasmic ribosomes)
Inhibits initiation, binds between P- & A- sites
Eukaryotic (Pf cytoplasmic ribosomes)
Inhibits initation & elongation, multiple binding sites
Eukaryotic (Pf cytoplasmic ribosomes)
Inhibits elongation, binds A-site
Eukaryotic (Pf cytoplasmic ribosomes)
Inhibits elongation, binds E-site
Eukaryotic (Pf cytoplasmic ribosomes)
Inhibits elongation on re-initiating ribosomes, binds A-site
Eukaryotic (Pf cytoplasmic ribosomes)
Inhibits elongation, binds E-site
Eukaryotic (Pf cytoplasmic ribosomes)
Inhibits elongation, binds A-site
Inhibits glutamyl-prolyl-tRNA synthetase
Prokaryotic (Pf mitochondrial & apicoplast ribosomes)
Inhibits initiation & elongation
Half-maximal effective concentrations (nM) determined in P. falciparum growth inhibition assay
72H growth inhibition EC50 (nM)
72H growth inhibition EC50 (nM)
The Plasmodium falciparum in vitro translation assay measures activity of cytoplasmic ribosomes
Importantly, all of the eukaryotic ribosome-specific inhibitors, which therefore should inhibit only P. falciparum cytoplasmic and not apicoplast or mitochondrial ribosomes, displayed inhibition in the PfIVT assay, with several achieving complete or near complete blocking of translation (suramin, anisomycin, lactimidomycin, nagilactone C) (Fig. 2). In addition, the prokaryotic ribosome-specific inhibitor thiostrepton did not inhibit the PfIVT assay at any concentration tested, despite inhibiting the S35 assay at concentrations above its determined EC50 (Fig. 3). Thiostrepton is known to have multiple targets apart from ribosomes in eukaryotes and has been shown to induce an ER stress response with a phenotype similar to thapsigargin, which likely accounts for its activity in the S35 assay [36–38].
The S35 incorporation assay is not a reliable indicator of direct translation inhibition
Although it is well documented in other model systems (i.e. yeast) that the S35-radiolabelled amino acid incorporation assay is an indirect measure of translation and can, as such, generate many misleading artifacts, this has not yet been characterized carefully with respect to Plasmodium spp. [39, 40]. Despite this, several studies in Plasmodium have relied on this indirect measure as a primary readout of translation [9, 41]. To address this and further determine the specificity of the PfIVT assay relative to the S35 uptake assay, a panel of small molecules that are known to inhibit cellular processes other than translation were tested (Tables 2 and 3). Not surprisingly, actinomycin D, an inhibitor of transcription targeting RNA Polymerase II, and the SERCA inhibitor thapsigargin both exhibited strong inhibition in the S35 incorporation assay, but had no effect in the PfIVT assay (Fig. 3). Tubercidin, an adenosine mimetic, had a modest inhibitory effect on S35 incorporation, but, again, negligible effect in the PfIVT assay (Fig. 3). These data confirm that the PfIVT assay directly measures translation, and highlight the lack of translation specificity of the S35 incorporation assay.
Analysis of clinically-approved anti-malarials reveals that none, including mefloquine, inhibit the 80S ribosome
Also included were several drugs (clinical and pre-clinical) that have recently been reported to inhibit translation (Table 3) [7–9]. MMV008270 was a moderate inhibitor of the S35 incorporation assay, while mefloquine and DDD107498 robustly inhibited S35 incorporation (Fig. 4). Strikingly, while DDD107498 and MMV008270 inhibited the PfIVT assay, mefloquine failed to do so (Fig. 4). Interestingly, MMV008270 was an exceptionally effective inhibitor of translation in the PfIVT assay at all concentrations tested (Fig. 4). These data reveal that mefloquine has recently been mischaracterized as a ribosome inhibitor through use of the S35 incorporation assay, when it does not, in fact, directly inhibit translation .
This work presents an extensive dissection and validation of the whole-cell extract-derived PfIVT assay, the only reported direct measure of P. falciparum translation to date. Through probing the assay with numerous small molecule inhibitors of translation, exhibiting a diversity of binding sites and mechanisms of action, as well as a variety of well-characterized tool compounds inhibiting non-translational pathways, it is demonstrated that the PfIVT assay specifically measures P. falciparum cytoplasmic ribosome activity. In vitro translation extracts are inherently difficult to make, and even more so for an intraerythrocytic parasite. However, when subjected to stringent quality control and careful optimization, the PfIVT assay reliably and specifically identifies inhibitors of translation initiation and elongation, as well as inhibitors of non-ribosomal proteins necessary for translation, such as tRNA synthetase.
The PfIVT assay is particularly valuable to the study of P. falciparum translation as a direct measure of translation, as opposed to the indirect measures to which the field has historically been constrained, such as incorporation of radiolabelled amino acids in vivo. Importantly, these data show the PfIVT assay is significantly more specific, and in some cases more sensitive, than S35-radiolabel incorporation in identifying small molecule inhibitors of translation. Indeed, the PfIVT assay specifically identified all eukaryotic translation inhibitors tested, while S35-radiolabel incorporation was prone to false-positives. This work shows that none of the clinically approved anti-malarials tested are inhibitors of translation, emphasizing the potential for translation as a useful therapeutic target, as there is unlikely to be pre-existing mechanism-specific resistance to any identified candidates resulting from use of these drugs. It is notable that mefloquine, in contrast to other previously reported translation inhibitors, did not exhibit any inhibitory activity. Mefloquine was likely mischaracterized as an 80S ribosome inhibitor through a combination of non-specific inhibition of S35 incorporation, as well as artifacts arising from cryo-EM structures obtained under the non-physiologic condition of 10 mM magnesium—well above the ~ 4 mM magnesium found herein to be optimal for translation, as well as the physiologic magnesium concentrations noted in other eukaryotes and E. coli (Fig. 1) [9, 44–46].
While the PfIVT assay exhibits clear benefits over existing methodologies for the study of P. falciparum translation, it is acknowledged that the technique has several limitations. As is the case with in vitro translation systems in other organisms, the current assay is likely biased toward the study of non-cap-dependent initiation and elongation. Uncapped mRNA were utilized in this study to focus specifically on activity of the 80S ribosome itself, rather than the cap-recognition apparatus. It is possible that utilization of capped mRNA in future studies would facilitate interrogation of cap-dependent translation initiation. Likewise, there are few characterized pharmacologic inhibitors of eukaryotic translation termination, none of which are currently commercially available; thus, the PfIVT system, as described, may not be sensitive to all specific inhibitors of translation termination. Additionally, some translation inhibitors, such as homoharringtonine, demonstrated greater potency in the S35 incorporation and growth inhibition assays than in PfIVT. Such variation between the two assays may suggest off-target effects of these drugs, or differences between whole living cells and cellular extracts.
Determining the true molecular targets of anti-malarials is critical to improved therapeutic development. Exploiting differences between P. falciparum and mammalian ribosomes remains a promising avenue, as evidenced by the potent and discriminating drug DDD107498. Here, it is shown that orthogonal biochemical assays may be used to test hypotheses generated by structural data and cell-based assessments. This investigation of mefloquine reaffirms that direct functional measurements of drug activity are critical to identifying the genuine molecular targets of drugs. Importantly, this work demonstrates that the PfIVT assay is a uniquely direct measure of P. falciparum translation that can be used to facilitate a better understanding of the specifics of P. falciparum protein synthesis, with potentially great consequences for anti-malarial therapeutic development.
CMS, VA, VEG, and JLD conceived and designed the study, drafted, and revised the paper and its figures. CMS, VEG, and VA performed and analysed parasite growth experiments. CMS and VEG performed and analysed S35 incorporation assays. CMS and VA performed and analysed in vitro translation assays. CMS performed and analysed magnesium assays. All authors read and approved the final manuscript.
Dr. Phil Rosenthal is thanked for providing stocks of several antimalarial drugs. We thank Wesley Wu for assistance and advice.
The authors declare that they have no competing interests.
Availability of data and materials
Consent for publication
Ethics approval and consent to participate
This work was funded by the Chan Zuckerberg Biohub (http://www.czbiohub.org). VEG is supported by the NIH T32 training grant (T32GM007810_32), CMS is supported by the UCSF Discovery Fellows Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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