Identification of Plasmodium falciparum specific translation inhibitors from the MMV Malaria Box using a high throughput in vitro translation screen
© Ahyong et al. 2016
Received: 14 January 2016
Accepted: 11 March 2016
Published: 17 March 2016
A major goal in the search for new anti-malarial compounds is to identify new mechanisms of action or new molecular targets. While cell-based, growth inhibition-based screening have enjoyed tremendous success, an alternative approach is to specifically assay a given pathway or essential cellular process.
Here, this work describes the development of a plate-based, in vitro luciferase assay to probe for inhibitors specific to protein synthesis in Plasmodium falciparum through the use of an in vitro translation system derived from the parasite.
Using the Medicines for Malaria Venture’s Malaria Box as a pilot, 400 bioactive compounds with minimal human cytotoxicity profiles were screened, identifying eight compounds that displayed greater potency against the P. falciparum translation machinery relative to a mammalian translation system. Dose–response curves were determined in both translation systems to further characterize the top hit compound (MMV008270).
This assay will be useful not only in future anti-malarial screening efforts but also in the investigation of P. falciparum protein synthesis and essential processes in P. falciparum biology.
Identifying new anti-malarials with novel mechanisms of action is a key goal in the fight to eradicate malaria worldwide . A common and very successful strategy relies on screening in vitro cultures of Plasmodium falciparum against large compound collections and assaying for growth inhibition in a ‘top-down’ approach to drug discovery. Using this type of approach can result in the subsequent identification of drug targets by selection of resistant strains and whole genome sequencing of these resistant strains to identify mutations (i.e., single nucleotide polymorphisms, copy number variants, insertions/deletions) that confer resistance [2–4]. However, one challenge with this approach is the high likelihood of encountering new compounds associated with targets that have previously been exploited as opposed to identifying new mechanisms of action. This has certainly been the case in recent instances, in which multiple groups have identified diverse chemical compounds that all have the same molecular determinants of resistance, such as PfATP4, and quite possibly the same mechanism of action [2, 5, 6] .
The complementary strategy is to narrow the search criteria by assaying for activity against a specific biological function or pathway. For example, this approach was used to identify a specific inhibitor of PfIspD, an enzyme essential for isoprenoid synthesis, by counter screening with growth media supplemented with isopentenyl pyrophosphate (IPP), thus narrowing hits to only those active against apicoplast targets (such as isoprenoid enzymes) . Facilitating these efforts, the freely available Medicines for Malaria Venture (MMV) Malaria Box has been a welcome resource, providing biologically active compounds with unknown targets and mechanisms of action . The library contains 400 chemically diverse compounds that are commercially available and pre-screened for activity in the blood stages of P. falciparum with minimal human cytotoxicity.
Among the possible pathways that can be functionally assayed, protein synthesis represents an attractive target, given its absolutely essential nature. Indeed, despite the fact that P. falciparum is a eukaryotic organism, there are ample differences between the P. falciparum and mammalian ribosomes that could be plausibly exploited [8, 9]. In fact, precedence for this type of inhibition of protein synthesis was exemplified in the discovery of the sordarin class of natural products which selectively inhibits fungal protein synthesis by inhibiting the yeast eukaryotic elongation factor 2 . In a similar manner, a potent new compound, DDD107498, was reported to specifically inhibit P. falciparum protein synthesis by blocking activity of the P. falciparum translation eukaryotic elongation factor 2 . Here, this study reports the use of a P. falciparum in vitro translation assay, amenable to plate-based screening, to identify inhibitors of P. falciparum translation present in the Malaria Box.
Plasmodium falciparum culturing
W2 strains were maintained in HYPERFlasks (Corning, Corning, NY, USA) in 500 mL RPMIc [RPMI 1640 media supplemented with 0.25 % Albumax II (GIBCO, Grand Island, NY, USA), 2 g/L sodium bicarbonate, 25 mM HEPES (pH 7.4), 0.1 mM hypoxanthine, and 50 ug/L gentamycin] in a 37 °C, 5 % O2, 5 % CO2 incubator in 2 % haematocrit (HC). Cells were synchronized with 5 % sorbitol treatment for two generations to achieve high synchronicity.
Harvesting cell pellets
One liter parasite cultures grown in two 500 mL HYPER flasks were harvested in the late trophozoite stage at approximately 15 % parasitaemia by centrifugation for 5 min at 1500×g at room temperature and 0.06 % final saponin in Buffer A (20 mM HEPES pH 8.0, 2 mM Mg(OAc)2, 120 mM KOAc). Saponin lysed pellets were centrifuged at 4 °C 10,000×g for 10 min and washed once with ice-cold Buffer A. The pellet was resuspended in 2 mL of Buffer B2 [20 mM HEPES pH 8.0, 100 mM KOAc, 0.75 mM Mg(OAC)2, 2 mM DTT, 20 % glycerol, 1× protease inhibitor cocktail (Roche)], flash frozen, and stored in −80 °C freezer until the sample was ready to homogenize.
Homogenization of cell pellets
Frozen pellets were thawed on ice and added to a 3-mL luer lock syringe, locked onto a pre-chilled cell homogenizer (Isobiotec, Germany) on ice and passed between two syringes 20 times. Lysate was centrifuged at 4 °C 16,000×g for 10 min and the supernatant was stored at −80 °C.
In vitro translation assay
Plasmodium in vitro translation reactions were carried out v-bottom 96-well PCR plates (USA Scientific, Ocala, FL, USA) and sealed with adhesive aluminum foil plate seals (Beckman Coulter, Indianapolis, IN, USA) with the following components in 20 μL: 16 μL lysate, 1 μg T7 transcribed firefly luciferase mRNA, 10 µM amino acid mixture, 20 mM HEPES/KOH pH 8.0, 75 mM KOAc, 2 mM Mg(OAc)2, 2 mM DTT, 0.5 mM ATP, 0.1 mM GTP, 20 mM creatine phosphate, 0.2 μg/μl creatine kinase for 0.5–1.5 h at 37 °C. All liquid dispensing was performed using Rainin E4 12-channel electronic pipettes (Rainin Instruments, Oakland, CA, USA). After incubation, the reactions were quenched with a final concentration of 5 µM cycloheximide. Samples were transferred to a 96-well LUMITRAC 200 white immunology plate (Greiner Bio-One, Monroe, NC, USA). Reactions were assayed using the Promega GloMax-Multi + microplate reader with a three-second delay and ten-second integration after addition of 100 μL 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).
Rabbit reticulocyte (Retic Lysate IVT Kit, Thermo Fisher Scientific, Waltham, MA, USA) in vitro translation assays were carried out with the following components in 20 μL: 0.5 μL 20× translation mix minus methionine, 0.5 μL 20× translation mix minus leucine, 1 μg T7 transcribed firefly luciferase mRNA, and 18 μL reticulocyte lysate for 5–20 min at 37 °C. After incubation, the reactions were quenched with 5 µM cycloheximide and assayed in the same manner as the Plasmodium lysates.
The open-access Malaria Box was obtained from MMV. Initial screens were performed at 1 μM final drug concentration in v-bottom 96-well PCR plates (USA Scientific, Ocala, FL, USA) and sealed with adhesive aluminum foil plate seals (Beckman Coulter, Indianapolis, IN, USA). The 20 μL reaction contained the following components in 20 μL: 14 μL lysate, 1 μg T7 transcribed firefly luciferase mRNA, 10 µM amino acid mixture, 20 mM HEPES/KOH pH 8.0, 75 mM KOAc, 2 mM Mg(OAc)2, 2 mM DTT, 0.5 mM ATP, 0.1 mM GTP, 20 mM creatine phosphate, 0.2 μg/μl creatine kinase. After the 1.5 h incubation, samples were immediately quenched using 2μL of 50 μM cycloheximide stop solution and then transferred to the Lumitrac assay plate using 12-channel electronic pipettes and assayed in the same manner described in the above in vitro translation assay. IC50 values were determined with a constant 2.5 % DMSO, using a 12-point 1:3 titration starting at 250 μM. Data were normalized to background and DMSO-only controls.
Development of a high-throughput, malaria-specific, in vitro translation assay
Screening the MMV Malaria Box for translational inhibitors
Eight top hit compounds
Alternative compound number
Average Pf IVT
CHEMBL IC50 in uM
CHEMBL IC50 in human fibroblast (MRC-5) cells to measure toxicity
This work presents a novel high-throughput luciferase assay that enables the discovery of compounds inhibiting eukaryotic protein synthesis in P. falciparum. This cell-free system is sensitive to the known translation inhibitor cycloheximide, whereas anti-malarials with no known effect on translation, such as chloroquine and DHA, do not exhibit inhibitory activity in the assay. Utilizing this novel assay resulted in the identification of eight compounds that are inhibitors of P. falciparum translation, and more effectively inhibit P. falciparum than mammalian in vitro translation. Considering that this assay is based on a crude extract that naturally has more variability than a pure enzyme assay, additional secondary screening (such as IC curves in this case) is required to establish their validity. Of these eight compounds, further characterization of the top hit, MMV008270, showed 1.9× higher activity against P. falciparum translation over a general eukaryotic translation system. While a 1.9× difference is likely to be too narrow for serious consideration as a lead molecule, it provides a starting point for further structure activity relationship studies with the goal of widening the gap between P. falciparum and mammalian inhibition. Further, target validation by selection for resistance may aid in determining which component of the ribosomal machinery is the target of this compound. It is likely that multiple components of the ribosome could be targeted simultaneously to yield a synergistic effect.
The assay developed here has the potential to be a powerful tool for additional translation-specific drug screens as well as answering important questions of basic Plasmodium biology. For example, future experiments using this streamlined, multi-well, plate-friendly assay will allow for the further investigation of cis-acting determinants of translational efficiency that were identified in a previous study . In total, this work presents a valuable molecular tool for probing an essential process in P. falciparum biology.
Availability of supporting data
The data set supporting the results of this article will be freely available in the CHEMBL MMV repository (https://www.ebi.ac.uk/chembl/malaria/).
VA and JLD conceived and designed the study. VA, CMS, KEL, JNW, JD, and JLD performed experiments and analysed the data. VA, CMS and JLD drafted or revised the paper. All authors read and approved the final manuscript.
We thank members of the DeRisi Laboratory for thoughtful discussions and culturing assistance, especially Daniel Ebert, Wesley Wu, Florence Caro, and Ellen Yeh. JLD and this work were supported by the Howard Hughes Medical Institute. VA and JNW were supported by the NIH T32 Grant GM007810. CMS was supported by the UCSF Discovery Fellows Program. KEL and JD were supported by the HHMI Extraordinary Research Opportunities Program (ExROP).
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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