Publications by authors named "Jake Baum"

99 Publications

Correction: Single-molecule nanopore sensing of actin dynamics and drug binding.

Chem Sci 2020 Jul 23;11(30):8036-8038. Epub 2020 Jul 23.

Department of Chemistry, Imperial College London, Molecular Sciences Research Hub White City Campus 80 Wood Lane W12 0BZ UK

[This corrects the article DOI: 10.1039/C9SC05710B.].
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http://dx.doi.org/10.1039/d0sc90132fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8163301PMC
July 2020

A single-cell atlas of Plasmodium falciparum transmission through the mosquito.

Nat Commun 2021 05 27;12(1):3196. Epub 2021 May 27.

Parasites and Microbes Programme, Wellcome Sanger Institute, Hinxton, UK.

Malaria parasites have a complex life cycle featuring diverse developmental strategies, each uniquely adapted to navigate specific host environments. Here we use single-cell transcriptomics to illuminate gene usage across the transmission cycle of the most virulent agent of human malaria - Plasmodium falciparum. We reveal developmental trajectories associated with the colonization of the mosquito midgut and salivary glands and elucidate the transcriptional signatures of each transmissible stage. Additionally, we identify both conserved and non-conserved gene usage between human and rodent parasites, which point to both essential mechanisms in malaria transmission and species-specific adaptations potentially linked to host tropism. Together, the data presented here, which are made freely available via an interactive website, provide a fine-grained atlas that enables intensive investigation of the P. falciparum transcriptional journey. As well as providing insights into gene function across the transmission cycle, the atlas opens the door for identification of drug and vaccine targets to stop malaria transmission and thereby prevent disease.
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http://dx.doi.org/10.1038/s41467-021-23434-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8159942PMC
May 2021

Identification and characterisation of a phospholipid scramblase in the malaria parasite Plasmodium falciparum.

Mol Biochem Parasitol 2021 May 8;243:111374. Epub 2021 May 8.

Department of Life Sciences, Imperial College London, Sir Alexander Fleming Building, Exhibition Road, South Kensington, London, UK. Electronic address:

Recent studies highlight the emerging role of lipids as important messengers in malaria parasite biology. In an attempt to identify interacting proteins and regulators of these dynamic and versatile molecules, we hypothesised the involvement of phospholipid translocases and their substrates in the infection of the host erythrocyte by the malaria parasite Plasmodium spp. Here, using a data base searching approach of the Plasmodium Genomics Resources (www.plasmodb.org), we have identified a putative phospholipid (PL) scramblase in P. falciparum (PfPLSCR) that is conserved across the genus and in closely related unicellular algae. By reconstituting recombinant PfPLSCR into liposomes, we demonstrate metal ion dependent PL translocase activity and substrate preference, confirming PfPLSCR as a bona fide scramblase. We show that PfPLSCR is expressed during asexual and sexual parasite development, localising to different membranous compartments of the parasite throughout the intra-erythrocytic life cycle. Two different gene knockout approaches, however, suggest that PfPLSCR is not essential for erythrocyte invasion and asexual parasite development, pointing towards a possible role in other stages of the parasite life cycle.
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http://dx.doi.org/10.1016/j.molbiopara.2021.111374DOI Listing
May 2021

Repositioning and Characterization of 1-(Pyridin-4-yl)pyrrolidin-2-one Derivatives as Cytoplasmic Prolyl-tRNA Synthetase Inhibitors.

ACS Infect Dis 2021 Jun 30;7(6):1680-1689. Epub 2021 Apr 30.

Medicines for Malaria Venture, ICC, Route de Pré-Bois 20, 1215 Geneva, Switzerland.

Prolyl-tRNA synthetase (PRS) is a clinically validated antimalarial target. Screening of a set of PRS ATP-site binders, initially designed for human indications, led to identification of 1-(pyridin-4-yl)pyrrolidin-2-one derivatives representing a novel antimalarial scaffold. Evidence designates cytoplasmic PRS as the drug target. The frontrunner and its active enantiomer exhibited low-double-digit nanomolar activity against resistant () laboratory strains and development of liver schizonts. No cross-resistance with strains resistant to other known antimalarials was noted. In addition, a similar level of growth inhibition was observed against clinical field isolates of and . The slow killing profile and the relative high propensity to develop resistance (minimum inoculum resistance of 8 × 10 parasites at a selection pressure of 3 × IC) constitute unfavorable features for treatment of malaria. However, potent blood stage and antischizontal activity are compelling for causal prophylaxis which does not require fast onset of action. Achieving sufficient on-target selectivity appears to be particularly challenging and should be the primary focus during the next steps of optimization of this chemical series. Encouraging preliminary off-target profile and oral efficacy in a humanized murine model of malaria allowed us to conclude that 1-(pyridin-4-yl)pyrrolidin-2-one derivatives represent a promising starting point for the identification of novel antimalarial prophylactic agents that selectively target PRS.
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http://dx.doi.org/10.1021/acsinfecdis.1c00020DOI Listing
June 2021

Novel Antimalarial Tetrazoles and Amides Active against the Hemoglobin Degradation Pathway in .

J Med Chem 2021 03 23;64(5):2739-2761. Epub 2021 Feb 23.

Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, United States.

Malaria control programs continue to be threatened by drug resistance. To identify new antimalarials, we conducted a phenotypic screen and identified a novel tetrazole-based series that shows fast-kill kinetics and a relatively low propensity to develop high-level resistance. Preliminary structure-activity relationships were established including identification of a subseries of related amides with antiplasmodial activity. Assaying parasites with resistance to antimalarials led us to test whether the series had a similar mechanism of action to chloroquine (CQ). Treatment of synchronized parasites with active analogues revealed a pattern of intracellular inhibition of hemozoin (Hz) formation reminiscent of CQ's action. Drug selections yielded only modest resistance that was associated with amplification of the multidrug resistance gene 1 (). Thus, we have identified a novel chemical series that targets the historically druggable heme polymerization pathway and that can form the basis of future optimization efforts to develop a new malaria treatment.
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http://dx.doi.org/10.1021/acs.jmedchem.0c02022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7997635PMC
March 2021

The antimalarial efficacy and mechanism of resistance of the novel chemotype DDD01034957.

Sci Rep 2021 Jan 21;11(1):1888. Epub 2021 Jan 21.

Department of Infection Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, WC1E 7HT, UK.

New antimalarial therapeutics are needed to ensure that malaria cases continue to be driven down, as both emerging parasite resistance to frontline chemotherapies and mosquito resistance to current insecticides threaten control programmes. Plasmodium, the apicomplexan parasite responsible for malaria, causes disease pathology through repeated cycles of invasion and replication within host erythrocytes (the asexual cycle). Antimalarial drugs primarily target this cycle, seeking to reduce parasite burden within the host as fast as possible and to supress recrudescence for as long as possible. Intense phenotypic drug screening efforts have identified a number of promising new antimalarial molecules. Particularly important is the identification of compounds with new modes of action within the parasite to combat existing drug resistance and suitable for formulation of efficacious combination therapies. Here we detail the antimalarial properties of DDD01034957-a novel antimalarial molecule which is fast-acting and potent against drug resistant strains in vitro, shows activity in vivo, and possesses a resistance mechanism linked to the membrane transporter PfABCI3. These data support further medicinal chemistry lead-optimization of DDD01034957 as a novel antimalarial chemical class and provide new insights to further reduce in vivo metabolic clearance.
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http://dx.doi.org/10.1038/s41598-021-81343-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820608PMC
January 2021

Batch equalization with a generative adversarial network.

Bioinformatics 2020 12;36(Suppl_2):i875-i883

Google Research, 1600 Amphitheatre Parkway Mountain View, CA 94043.

Motivation: Advances in automation and imaging have made it possible to capture a large image dataset that spans multiple experimental batches of data. However, accurate biological comparison across the batches is challenged by batch-to-batch variation (i.e. batch effect) due to uncontrollable experimental noise (e.g. varying stain intensity or cell density). Previous approaches to minimize the batch effect have commonly focused on normalizing the low-dimensional image measurements such as an embedding generated by a neural network. However, normalization of the embedding could suffer from over-correction and alter true biological features (e.g. cell size) due to our limited ability to interpret the effect of the normalization on the embedding space. Although techniques like flat-field correction can be applied to normalize the image values directly, they are limited transformations that handle only simple artifacts due to batch effect.

Results: We present a neural network-based batch equalization method that can transfer images from one batch to another while preserving the biological phenotype. The equalization method is trained as a generative adversarial network (GAN), using the StarGAN architecture that has shown considerable ability in style transfer. After incorporating new objectives that disentangle batch effect from biological features, we show that the equalized images have less batch information and preserve the biological information. We also demonstrate that the same model training parameters can generalize to two dramatically different types of cells, indicating this approach could be broadly applicable.

Availability And Implementation: https://github.com/tensorflow/gan/tree/master/tensorflow_gan/examples/stargan.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btaa819DOI Listing
December 2020

Transmission of Artemisinin-Resistant Malaria Parasites to Mosquitoes under Antimalarial Drug Pressure.

Antimicrob Agents Chemother 2020 12 16;65(1). Epub 2020 Dec 16.

Department of Life Sciences, Imperial College London, London, United Kingdom

Resistance to artemisinin-based combination therapy (ACT) in the parasite is threatening to reverse recent gains in reducing global deaths from malaria. While resistance manifests as delayed parasite clearance in patients, the phenotype can only spread geographically via the sexual stages and mosquito transmission. In addition to their asexual killing properties, artemisinin and its derivatives sterilize sexual male gametocytes. Whether resistant parasites overcome this sterilizing effect has not, however, been fully tested. Here, we analyzed clinical isolates from the Greater Mekong Subregion, each demonstrating delayed clinical clearance and known resistance-associated polymorphisms in the (PfK13) gene. As well as demonstrating reduced asexual sensitivity to drug, certain PfK13 isolates demonstrated a marked reduction in sensitivity to artemisinin in an male gamete formation assay. Importantly, this same reduction in sensitivity was observed when the most resistant isolate was tested directly in mosquito feeds. These results indicate that, under artemisinin drug pressure, while sensitive parasites are blocked, resistant parasites continue transmission. This selective advantage for resistance transmission could favor acquisition of additional host-specificity or polymorphisms affecting partner drug sensitivity in mixed infections. Favored resistance transmission under ACT coverage could have profound implications for the spread of multidrug-resistant malaria beyond Southeast Asia.
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http://dx.doi.org/10.1128/AAC.00898-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7927852PMC
December 2020

Actomyosin forces and the energetics of red blood cell invasion by the malaria parasite Plasmodium falciparum.

PLoS Pathog 2020 10 26;16(10):e1009007. Epub 2020 Oct 26.

Department of Life Sciences, Imperial College London, South Kensington, London, United Kingdom.

All symptoms of malaria disease are associated with the asexual blood stages of development, involving cycles of red blood cell (RBC) invasion and egress by the Plasmodium spp. merozoite. Merozoite invasion is rapid and is actively powered by a parasite actomyosin motor. The current accepted model for actomyosin force generation envisages arrays of parasite myosins, pushing against short actin filaments connected to the external milieu that drive the merozoite forwards into the RBC. In Plasmodium falciparum, the most virulent human malaria species, Myosin A (PfMyoA) is critical for parasite replication. However, the precise function of PfMyoA in invasion, its regulation, the role of other myosins and overall energetics of invasion remain unclear. Here, we developed a conditional mutagenesis strategy combined with live video microscopy to probe PfMyoA function and that of the auxiliary motor PfMyoB in invasion. By imaging conditional mutants with increasing defects in force production, based on disruption to a key PfMyoA phospho-regulation site, the absence of the PfMyoA essential light chain, or complete motor absence, we define three distinct stages of incomplete RBC invasion. These three defects reveal three energetic barriers to successful entry: RBC deformation (pre-entry), mid-invasion initiation, and completion of internalisation, each requiring an active parasite motor. In defining distinct energetic barriers to invasion, these data illuminate the mechanical challenges faced in this remarkable process of protozoan parasitism, highlighting distinct myosin functions and identifying potential targets for preventing malaria pathogenesis.
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http://dx.doi.org/10.1371/journal.ppat.1009007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7644091PMC
October 2020

Full-length myosin A and essential light chain PfELC structures provide new anti-malarial targets.

Elife 2020 10 13;9. Epub 2020 Oct 13.

Structural Motility, Institut Curie, Paris Université Sciences et Lettres, Sorbonne Université, CNRS UMR144, Paris, France.

Parasites from the genus Plasmodium are the causative agents of malaria. The mobility, infectivity, and ultimately pathogenesis of rely on a macromolecular complex, called the glideosome. At the core of the glideosome is an essential and divergent Myosin A motor (PfMyoA), a first order drug target against malaria. Here, we present the full-length structure of PfMyoA in two states of its motor cycle. We report novel interactions that are essential for motor priming and the mode of recognition of its two light chains (PfELC and MTIP) by two degenerate IQ motifs. Kinetic and motility assays using PfMyoA variants, along with molecular dynamics, demonstrate how specific priming and atypical sequence adaptations tune the motor's mechano-chemical properties. Supported by evidence for an essential role of the PfELC in malaria pathogenesis, these structures provide a blueprint for the design of future anti-malarials targeting both the glideosome motor and its regulatory elements.
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http://dx.doi.org/10.7554/eLife.60581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7553781PMC
October 2020

A machine learning approach to define antimalarial drug action from heterogeneous cell-based screens.

Sci Adv 2020 Sep 25;6(39). Epub 2020 Sep 25.

Department of Life Sciences, Imperial College London, London, UK.

Drug resistance threatens the effective prevention and treatment of an ever-increasing range of human infections. This highlights an urgent need for new and improved drugs with novel mechanisms of action to avoid cross-resistance. Current cell-based drug screens are, however, restricted to binary live/dead readouts with no provision for mechanism of action prediction. Machine learning methods are increasingly being used to improve information extraction from imaging data. These methods, however, work poorly with heterogeneous cellular phenotypes and generally require time-consuming human-led training. We have developed a semi-supervised machine learning approach, combining human- and machine-labeled training data from mixed human malaria parasite cultures. Designed for high-throughput and high-resolution screening, our semi-supervised approach is robust to natural parasite morphological heterogeneity and correctly orders parasite developmental stages. Our approach also reproducibly detects and clusters drug-induced morphological outliers by mechanism of action, demonstrating the potential power of machine learning for accelerating cell-based drug discovery.
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http://dx.doi.org/10.1126/sciadv.aba9338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7518791PMC
September 2020

Synthesis and Structure-Activity Relationship of Dual-Stage Antimalarial Pyrazolo[3,4-]pyridines.

J Med Chem 2020 10 1;63(20):11902-11919. Epub 2020 Oct 1.

Medicines for Malaria Venture (MMV), P.O. Box 1826, 20, Route de Pré-Bois, Geneva 1215, Switzerland.

Malaria remains one of the most deadly infectious diseases, causing hundreds of thousands of deaths each year, primarily in young children and pregnant mothers. Here, we report the discovery and derivatization of a series of pyrazolo[3,4-]pyridines targeting , the deadliest species of the malaria parasite. Hit compounds in this series display sub-micromolar activity against the intraerythrocytic stage of the parasite as well as little to no toxicity against the human fibroblast BJ and liver HepG2 cell lines. In addition, our hit compounds show good activity against the liver stage of the parasite but little activity against the gametocyte stage. Parasitological profiles, including rate of killing, docking, and molecular dynamics studies, suggest that our compounds may target the Q binding site of cytochrome .
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http://dx.doi.org/10.1021/acs.jmedchem.0c01152DOI Listing
October 2020

Structure-Activity Studies of Truncated Latrunculin Analogues with Antimalarial Activity.

ChemMedChem 2021 Feb 10;16(4):679-693. Epub 2020 Nov 10.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia.

Malarial parasites employ actin dynamics for motility, and any disruption to these dynamics renders the parasites unable to effectively establish infection. Therefore, actin presents a potential target for malarial drug discovery, and naturally occurring actin inhibitors such as latrunculins are a promising starting point. However, the limited availability of the natural product and the laborious route for synthesis of latrunculins have hindered their potential development as drug candidates. In this regard, we recently described novel truncated latrunculins, with superior actin binding potency and selectivity towards P. falciparum actin than the canonical latrunculin B. In this paper, we further explore the truncated latrunculin core to summarize the SAR for inhibition of malaria motility. This study helps further understand the binding pattern of these analogues in order to develop them as drug candidates for malaria.
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http://dx.doi.org/10.1002/cmdc.202000399DOI Listing
February 2021

Conditional expression of PfAP2-G for controlled massive sexual conversion in .

Sci Adv 2020 Jun 10;6(24):eaaz5057. Epub 2020 Jun 10.

ISGlobal, Hospital Clinic-Universitat de Barcelona, Barcelona 08036, Catalonia, Spain.

Malaria transmission requires that some asexual parasites convert into sexual forms termed gametocytes. The initial stages of sexual development, including sexually committed schizonts and sexual rings, remain poorly characterized, mainly because they are morphologically identical to their asexual counterparts and only a small subset of parasites undergo sexual development. Here, we describe a system for controlled sexual conversion in the human malaria parasite , based on conditional expression of the PfAP2-G transcription factor. Using this system, ~90 percent of the parasites converted into sexual forms upon induction, enabling the characterization of committed and early sexual stages without further purification. We characterized sexually committed schizonts and sexual rings at the transcriptomic and phenotypic levels, which revealed down-regulation of genes involved in solute transport upon sexual commitment, among other findings. The new inducible lines will facilitate the study of early sexual stages at additional levels, including multiomic characterization and drug susceptibility assays.
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http://dx.doi.org/10.1126/sciadv.aaz5057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7286680PMC
June 2020

Peptide Probes for MyoA Tail Interacting Protein (MTIP): Exploring the Druggability of the Malaria Parasite Motor Complex.

ACS Chem Biol 2020 06 13;15(6):1313-1320. Epub 2020 May 13.

Department of Life Sciences, Imperial College, London SW7 2AZ, United Kingdom.

Malaria remains an endemic tropical disease, and the emergence of parasites resistant to current front-line medicines means that new therapeutic targets are required. The glideosome is a multiprotein complex thought to be essential for efficient host red blood cell invasion. At its core is a myosin motor, Myosin A (MyoA), which provides most of the force required for parasite invasion. Here, we report the design and development of improved peptide-based probes for the anchor point of MyoA, the MyoA tail interacting protein (MTIP). These probes combine low nanomolar binding affinity with significantly enhanced cell penetration and demonstrable competitive target engagement with native MTIP through a combination of Western blot and chemical proteomics. These results provide new insights into the potential druggability of the MTIP/MyoA interaction and a basis for the future design of inhibitors.
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http://dx.doi.org/10.1021/acschembio.0c00328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7309260PMC
June 2020

A Biosynthetic Platform for Antimalarial Drug Discovery.

Antimicrob Agents Chemother 2020 04 21;64(5). Epub 2020 Apr 21.

Department of Life Sciences, Imperial College London, London, United Kingdom

Advances in synthetic biology have enabled the production of a variety of compounds using bacteria as a vehicle for complex compound biosynthesis. Violacein, a naturally occurring indole pigment with antibiotic properties, can be biosynthetically engineered in expressing its nonnative synthesis pathway. To explore whether this synthetic biosynthesis platform could be used for drug discovery, here we have screened bacterially derived violacein against the main causative agent of human malaria, We show the antiparasitic activity of bacterially derived violacein against the 3D7 laboratory reference strain as well as drug-sensitive and -resistant patient isolates, confirming the potential utility of this drug as an antimalarial agent. We then screen a biosynthetic series of violacein derivatives against growth. The varied activity of each derivative against asexual parasite growth points to the need to further develop violacein as an antimalarial. Towards defining its mode of action, we show that biosynthetic violacein affects the parasite actin cytoskeleton, resulting in an accumulation of actin signal that is independent of actin polymerization. This activity points to a target that modulates actin behavior in the cell either in terms of its regulation or its folding. More broadly, our data show that bacterial synthetic biosynthesis could become a suitable platform for antimalarial drug discovery, with potential applications in future high-throughput drug screening with otherwise chemically intractable natural products.
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http://dx.doi.org/10.1128/AAC.02129-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7179595PMC
April 2020

Single-molecule nanopore sensing of actin dynamics and drug binding.

Chem Sci 2019 Dec 3;11(4):970-979. Epub 2019 Dec 3.

Department of Chemistry, Imperial College London, Molecular Sciences Research Hub White City Campus, 80 Wood Lane W12 0BZ UK

Actin is a key protein in the dynamic processes within the eukaryotic cell. To date, methods exploring the molecular state of actin are limited to insights gained from structural approaches, providing a snapshot of protein folding, or methods that require chemical modifications compromising actin monomer thermostability. Nanopore sensing permits label-free investigation of native proteins and is ideally suited to study proteins such as actin that require specialised buffers and cofactors. Using nanopores, we determined the state of actin at the macromolecular level (filamentous or globular) and in its monomeric form bound to inhibitors. We revealed urea-dependent and voltage-dependent transitional states and observed the unfolding process within which sub-populations of transient actin oligomers are visible. We detected, in real-time, filament-growth, and drug-binding at the single-molecule level demonstrating the promise of nanopore sensing for in-depth understanding of protein folding landscapes and for drug discovery.
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http://dx.doi.org/10.1039/c9sc05710bDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8146688PMC
December 2019

The effects of dyslipidaemia and cholesterol modulation on erythrocyte susceptibility to malaria parasite infection.

Malar J 2019 Nov 29;18(1):381. Epub 2019 Nov 29.

Department of Life Sciences, Imperial College London, Exhibition Road, South Kensington, London, SW7 2AZ, UK.

Background: Malaria disease commences when blood-stage parasites, called merozoites, invade human erythrocytes. Whilst the process of invasion is traditionally seen as being entirely merozoite-driven, emerging data suggests erythrocyte biophysical properties markedly influence invasion. Cholesterol is a major determinant of cell membrane biophysical properties demanding its interrogation as a potential mediator of resistance to merozoite invasion of the erythrocyte.

Methods: Biophysical measurements of erythrocyte deformability by flicker spectroscopy were used to assess changes in erythrocyte bending modulus on forced integration of cholesterol and how these artificial changes affect invasion by human Plasmodium falciparum merozoites. To validate these observations in a natural context, either murine Plasmodium berghei or human Plasmodium falciparum merozoites were tested for their ability to invade erythrocytes from a hypercholesterolaemic mouse model or human clinical erythrocyte samples deriving from patients with a range of serum cholesterol concentrations, respectively.

Results: Erythrocyte bending modulus (a measure of deformability) was shown to be markedly affected by artificial modulation of cholesterol content and negatively correlated with merozoite invasion efficiency. In an in vitro infection context, however, erythrocytes taken from hypercholesterolaemic mice or from human clinical samples with varying serum cholesterol levels showed little difference in their susceptibility to merozoite invasion. Explaining this, membrane cholesterol levels in both mouse and human hypercholesterolaemia erythrocytes were subsequently found to be no different from matched normal serum controls.

Conclusions: Based on these observations, serum cholesterol does not appear to impact on erythrocyte susceptibility to merozoite entry. Indeed, no relationship between serum cholesterol and cholesterol content of the erythrocyte is apparent. This work, nonetheless, suggests that native polymorphisms which do affect membrane lipid composition would be expected to affect parasite entry. This supports investigation of erythrocyte biophysical properties in endemic settings, which may yet identify naturally protective lipid-related polymorphisms.
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http://dx.doi.org/10.1186/s12936-019-3016-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6884832PMC
November 2019

Screen of traditional soup broths with reported antipyretic activity towards the discovery of potential antimalarials.

Arch Dis Child 2019 12;104(12):1138-1142

Department of Life Sciences, Imperial College London, London, UK

Objective: The global impact of artemisinin-based combination therapies on malaria-associated mortality and their origins in ancient Chinese medicine has heightened interest in the natural discovery of future antimalarials.

Methods: A double-blind study to identify potential ingredients with antimalarial activity from traditional remedies with reported antipyretic properties. Recipes of clear broths, passed down by tradition in families of diverse ethnic origin, were sourced by school children. Broths were then tested for their ability to arrest malaria parasite asexual growth or sexual stage development in vitro. Clear broth extract was incubated with in vitro cultures of asexual or mature sexual stage cultures and assayed for parasite viability after 72 hours.

Results: Of the 56 broths tested, 5 were found to give >50% in vitro growth inhibition against asexual blood stages, with 2 having comparable inhibition to that seen with dihydroartemisinin, a leading antimalarial. Four other broths were found to have >50% transmission blocking activity, preventing male parasite sexual stage development. After unblinding, two active broths were found to be from siblings from different classes, who had brought in the same vegetarian soup, demonstrating assay robustness.

Conclusions: This screening approach succeeded in finding broths with activity against malaria parasite growth, arising from complex vegetable and/or meat-based broths. This represented a successful child education exercise, in teaching about the interface between natural remedies, traditional medicine and evidence-based drug discovery.
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http://dx.doi.org/10.1136/archdischild-2019-317590DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6900245PMC
December 2019

Apicomplexan F-actin is required for efficient nuclear entry during host cell invasion.

EMBO Rep 2019 12 4;20(12):e48896. Epub 2019 Oct 4.

Wellcome Centre For Integrative Parasitology, Institute of Infection, Immunity & Inflammation, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, UK.

The obligate intracellular parasites Toxoplasma gondii and Plasmodium spp. invade host cells by injecting a protein complex into the membrane of the targeted cell that bridges the two cells through the assembly of a ring-like junction. This circular junction stretches while the parasites apply a traction force to pass through, a step that typically concurs with transient constriction of the parasite body. Here we analyse F-actin dynamics during host cell invasion. Super-resolution microscopy and real-time imaging highlighted an F-actin pool at the apex of pre-invading parasite, an F-actin ring at the junction area during invasion but also networks of perinuclear and posteriorly localised F-actin. Mutant parasites with dysfunctional acto-myosin showed significant decrease of junctional and perinuclear F-actin and are coincidently affected in nuclear passage through the junction. We propose that the F-actin machinery eases nuclear passage by stabilising the junction and pushing the nucleus through the constriction. Our analysis suggests that the junction opposes resistance to the passage of the parasite's nucleus and provides the first evidence for a dual contribution of actin-forces during host cell invasion by apicomplexan parasites.
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http://dx.doi.org/10.15252/embr.201948896DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6893294PMC
December 2019

Quantitative and rapid Plasmodium falciparum malaria diagnosis and artemisinin-resistance detection using a CMOS Lab-on-Chip platform.

Biosens Bioelectron 2019 Dec 7;145:111678. Epub 2019 Sep 7.

Centre for Bio-Inspired Technology, Department of Electrical and Electronic Engineering, Imperial College London, UK.

Early and accurate diagnosis of malaria and drug-resistance is essential to effective disease management. Available rapid malaria diagnostic tests present limitations in analytical sensitivity, drug-resistance testing and/or quantification. Conversely, diagnostic methods based on nucleic acid amplification stepped forwards owing to their high sensitivity, specificity and robustness. Nevertheless, these methods commonly rely on optical measurements and complex instrumentation which limit their applicability in resource-poor, point-of-care settings. This paper reports the specific, quantitative and fully-electronic detection of Plasmodium falciparum, the predominant malaria-causing parasite worldwide, using a Lab-on-Chip platform developed in-house. Furthermore, we demonstrate on-chip detection of C580Y, the most prevalent single-nucleotide polymorphism associated to artemisinin-resistant malaria. Real-time non-optical DNA sensing is facilitated using Ion-Sensitive Field-Effect Transistors, fabricated in unmodified complementary metal-oxide-semiconductor (CMOS) technology, coupled with loop-mediated isothermal amplification. This work holds significant potential for the development of a fully portable and quantitative malaria diagnostic that can be used as a rapid point-of-care test.
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http://dx.doi.org/10.1016/j.bios.2019.111678DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7224984PMC
December 2019

Structure-Activity Relationship Studies of a Novel Class of Transmission Blocking Antimalarials Targeting Male Gametes.

J Med Chem 2020 03 20;63(5):2240-2262. Epub 2019 Sep 20.

Department of Chemistry, Imperial College London, Molecular Sciences Research Hub, White City Campus, Wood Lane, London W12 0BZ, United Kingdom.

Malaria is still a leading cause of mortality among children in the developing world, and despite the immense progress made in reducing the global burden, further efforts are needed if eradication is to be achieved. In this context, targeting transmission is widely recognized as a necessary intervention toward that goal. After carrying out a screen to discover new transmission-blocking agents, herein we report our medicinal chemistry efforts to study the potential of the most robust hit, DDD01035881, as a male-gamete targeted compound. We reveal key structural features for the activity of this series and identify analogues with greater potency and improved metabolic stability. We believe this study lays the groundwork for further development of this series as a transmission blocking agent.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00898DOI Listing
March 2020

Genetic manipulation of cell line derived reticulocytes enables dissection of host malaria invasion requirements.

Nat Commun 2019 08 23;10(1):3806. Epub 2019 Aug 23.

Department of Life Sciences, Imperial College London, London, SW7 2AZ, United Kingdom.

Investigating the role that host erythrocyte proteins play in malaria infection is hampered by the genetic intractability of this anucleate cell. Here we report that reticulocytes derived through in vitro differentiation of an enucleation-competent immortalized erythroblast cell line (BEL-A) support both successful invasion and intracellular development of the malaria parasite Plasmodium falciparum. Using CRISPR-mediated gene knockout and subsequent complementation, we validate an essential role for the erythrocyte receptor basigin in P. falciparum invasion and demonstrate rescue of invasive susceptibility by receptor re-expression. Successful invasion of reticulocytes complemented with a truncated mutant excludes a functional role for the basigin cytoplasmic domain during invasion. Contrastingly, knockout of cyclophilin B, reported to participate in invasion and interact with basigin, did not impact invasive susceptibility of reticulocytes. These data establish the use of reticulocytes derived from immortalized erythroblasts as a powerful model system to explore hypotheses regarding host receptor requirements for P. falciparum invasion.
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http://dx.doi.org/10.1038/s41467-019-11790-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6707200PMC
August 2019

Plasmodium myosin A drives parasite invasion by an atypical force generating mechanism.

Nat Commun 2019 07 23;10(1):3286. Epub 2019 Jul 23.

Structural Motility, UMR 144 CNRS/Curie Institute, 26 rue d'ulm, 75258, Paris cedex 05, France.

Plasmodium parasites are obligate intracellular protozoa and causative agents of malaria, responsible for half a million deaths each year. The lifecycle progression of the parasite is reliant on cell motility, a process driven by myosin A, an unconventional single-headed class XIV molecular motor. Here we demonstrate that myosin A from Plasmodium falciparum (PfMyoA) is critical for red blood cell invasion. Further, using a combination of X-ray crystallography, kinetics, and in vitro motility assays, we elucidate the non-canonical interactions that drive this motor's function. We show that PfMyoA motor properties are tuned by heavy chain phosphorylation (Ser19), with unphosphorylated PfMyoA exhibiting enhanced ensemble force generation at the expense of speed. Regulated phosphorylation may therefore optimize PfMyoA for enhanced force generation during parasite invasion or for fast motility during dissemination. The three PfMyoA crystallographic structures presented here provide a blueprint for discovery of specific inhibitors designed to prevent parasite infection.
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http://dx.doi.org/10.1038/s41467-019-11120-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6650474PMC
July 2019

Design of a basigin-mimicking inhibitor targeting the malaria invasion protein RH5.

Proteins 2020 01 2;88(1):187-195. Epub 2019 Aug 2.

Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot, Israel.

Many human pathogens use host cell-surface receptors to attach and invade cells. Often, the host-pathogen interaction affinity is low, presenting opportunities to block invasion using a soluble, high-affinity mimic of the host protein. The Plasmodium falciparum reticulocyte-binding protein homolog 5 (RH5) provides an exciting candidate for mimicry: it is highly conserved and its moderate affinity binding to the human receptor basigin (K  ≥1 μM) is an essential step in erythrocyte invasion by this malaria parasite. We used deep mutational scanning of a soluble fragment of human basigin to systematically characterize point mutations that enhance basigin affinity for RH5 and then used Rosetta to design a variant within the sequence space of affinity-enhancing mutations. The resulting seven-mutation design exhibited 1900-fold higher affinity (K approximately 1 nM) for RH5 with a very slow binding off rate (0.23 h ) and reduced the effective Plasmodium growth-inhibitory concentration by at least 10-fold compared to human basigin. The design provides a favorable starting point for engineering on-rate improvements that are likely to be essential to reach therapeutically effective growth inhibition.
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http://dx.doi.org/10.1002/prot.25786DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6904230PMC
January 2020

Divergent roles for the RH5 complex components, CyRPA and RIPR in human-infective malaria parasites.

PLoS Pathog 2019 06 11;15(6):e1007809. Epub 2019 Jun 11.

Malaria Parasitology Laboratory, The Francis Crick Institute, London, United Kingdom.

Malaria is caused by Plasmodium parasites, which invade and replicate in erythrocytes. For Plasmodium falciparum, the major cause of severe malaria in humans, a heterotrimeric complex comprised of the secreted parasite proteins, PfCyRPA, PfRIPR and PfRH5 is essential for erythrocyte invasion, mediated by the interaction between PfRH5 and erythrocyte receptor basigin (BSG). However, whilst CyRPA and RIPR are present in most Plasmodium species, RH5 is found only in the small Laverania subgenus. Existence of a complex analogous to PfRH5-PfCyRPA-PfRIPR targeting BSG, and involvement of CyRPA and RIPR in invasion, however, has not been addressed in non-Laverania parasites. Here, we establish that unlike P. falciparum, P. knowlesi and P. vivax do not universally require BSG as a host cell invasion receptor. Although we show that both PkCyRPA and PkRIPR are essential for successful invasion of erythrocytes by P. knowlesi parasites in vitro, neither protein forms a complex with each other or with an RH5-like molecule. Instead, PkRIPR is part of a different trimeric protein complex whereas PkCyRPA appears to function without other parasite binding partners. It therefore appears that in the absence of RH5, outside of the Laverania subgenus, RIPR and CyRPA have different, independent functions crucial for parasite survival.
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http://dx.doi.org/10.1371/journal.ppat.1007809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6588255PMC
June 2019

Lysyl-tRNA synthetase as a drug target in malaria and cryptosporidiosis.

Proc Natl Acad Sci U S A 2019 04 20;116(14):7015-7020. Epub 2019 Mar 20.

Department of Life Sciences, Imperial College, South Kensington, SW7 2AZ London, United Kingdom.

Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage and in cell-culture studies. Target deconvolution in has shown that cladosporin inhibits lysyl-tRNA synthetase (KRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both KRS1 and KRS (KRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between KRS1 and KRS. This series of compounds inhibit KRS and and in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for KRS1 and KRS vs. (human) KRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.
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http://dx.doi.org/10.1073/pnas.1814685116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6452685PMC
April 2019

The antimalarial screening landscape-looking beyond the asexual blood stage.

Curr Opin Chem Biol 2019 06 12;50:1-9. Epub 2019 Mar 12.

Department of Life Sciences, Imperial College London, Sir Alexander Fleming Building, Exhibition Road, South Kensington, London SW7 2AZ, UK. Electronic address:

In recent years, the research agenda to tackle global morbidity and mortality from malaria disease has shifted towards innovation, in the hope that efforts at the frontiers of scientific research may re-invigorate gains made towards eradication. Discovery of new antimalarial drugs with novel chemotypes or modes of action lie at the heart of these efforts. There is a particular interest in drug candidates that target stages of the malaria parasite lifecycle beyond the symptomatic asexual blood stages. This is especially important given the spectre of emerging drug resistance to all current frontline antimalarials. One approach gaining increased interest is the potential of designing novel drugs that target parasite passage from infected individual to feeding mosquito and back again. Action of such therapeutics is geared much more at the population level rather than just concerned with the infected individual. The search for novel drugs active against these stages has been helped by improvements to in vitro culture of transmission and pre-erythrocytic parasite lifecycle stages, robotic automation and high content imaging, methodologies that permit the high-throughput screening (HTS) of compound libraries for drug discovery. Here, we review recent advances in the antimalarial screening landscape, focussed on transmission blocking as a key aim for drug-treatment campaigns of the future.
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http://dx.doi.org/10.1016/j.cbpa.2019.01.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6591700PMC
June 2019

Epistasis studies reveal redundancy among calcium-dependent protein kinases in motility and invasion of malaria parasites.

Nat Commun 2018 10 12;9(1):4248. Epub 2018 Oct 12.

Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, Geneva, CH-1211, Switzerland.

In malaria parasites, evolution of parasitism has been linked to functional optimisation. Despite this optimisation, most members of a calcium-dependent protein kinase (CDPK) family show genetic redundancy during erythrocytic proliferation. To identify relationships between phospho-signalling pathways, we here screen 294 genetic interactions among protein kinases in Plasmodium berghei. This reveals a synthetic negative interaction between a hypomorphic allele of the protein kinase G (PKG) and CDPK4 to control erythrocyte invasion which is conserved in P. falciparum. CDPK4 becomes critical when PKG-dependent calcium signals are attenuated to phosphorylate proteins important for the stability of the inner membrane complex, which serves as an anchor for the acto-myosin motor required for motility and invasion. Finally, we show that multiple kinases functionally complement CDPK4 during erythrocytic proliferation and transmission to the mosquito. This study reveals how CDPKs are wired within a stage-transcending signalling network to control motility and host cell invasion in malaria parasites.
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http://dx.doi.org/10.1038/s41467-018-06733-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6185908PMC
October 2018