A Microscale Human Liver Platform that Supports the Hepatic Stages of Plasmodium falciparum and vivax

The Plasmodium liver stage is an attractive target for the development of antimalarial drugs and vaccines, as it provides an opportunity to interrupt the life cycle of the parasite at a critical early stage. However, targeting the liver stage has been difficult. Undoubtedly, a major barrier has been the lack of robust, reliable, and reproducible in vitro liver-stage cultures. Here, we establish the liver stages for both Plasmodium falciparum and Plasmodium vivax in a microscale human liver platform composed of cryopreserved, micropatterned human primary hepatocytes surrounded by supportive stromal cells. Using this system, we have successfully recapitulated the full liver stage of P. falciparum, including the release of infected merozoites and infection of overlaid erythrocytes, as well as the establishment of small forms in late liver stages of P. vivax. Finally, we validate the potential of this platform as a tool for medium-throughput antimalarial drug screening and vaccine development.Bill & Melinda Gates Foundation (51066

Human iPSC-Derived Hepatocyte-like Cells Support Plasmodium Liver-Stage Infection In Vitro

SummaryMalaria eradication is a major goal in public health but is challenged by relapsing malaria species, expanding drug resistance, and the influence of host genetics on antimalarial drug efficacy. To overcome these hurdles, it is imperative to establish in vitro assays of liver-stage malaria for drug testing. Induced pluripotent stem cells (iPSC) potentially allow the assessment of donor-specific drug responses, and iPSC-derived hepatocyte-like cells (iHLCs) can facilitate the study of host genetics on host-pathogen interactions and the discovery of novel targets for antimalarial drug development. We establish in vitro liver-stage malaria infections in iHLCs using P. berghei, P. yoelii, P. falciparum, and P. vivax and show that differentiating cells acquire permissiveness to malaria infection at the hepatoblast stage. We also characterize antimalarial drug metabolism capabilities of iHLCs using prototypical antimalarial drugs and demonstrate that chemical maturation of iHLCs can improve their potential for antimalarial drug testing applications

Towards a Humanized Mouse Model of Liver Stage Malaria Using Ectopic Artificial Livers

The malaria liver stage is an attractive target for antimalarial development, and preclinical malaria models are essential for testing such candidates. Given ethical concerns and costs associated with non‐human primate models, humanized mouse models containing chimeric human livers offer a valuable alternative as small animal models of liver stage human malaria. The best available human liver chimeric mice rely on cellular transplantation into mice with genetically engineered liver injury, but these systems involve a long and variable humanization process, are expensive, and require the use of breeding-challenged mouse strains which are not widely accessible. We previously incorporated primary human hepatocytes into engineered polyethylene glycol (PEG)-based nanoporous human ectopic artificial livers (HEALs), implanted them in mice without liver injury, and rapidly generated human liver chimeric mice in a reproducible and scalable fashion. By re-designing the PEG scaffold to be macroporous, we demonstrate the facile fabrication of implantable porous HEALs that support liver stage human malaria (P. falciparum) infection in vitro, and also after implantation in mice with normal liver function, 60% of the time. This proof-of-concept study demonstrates the feasibility of applying a tissue engineering strategy towards the development of scalable preclinical models of liver stage malaria infection for future applications.Bill & Melinda Gates Foundation (Global Health Grant initiative (Global Health Grant: OPP1023607))National Cancer Institute (U.S.) (Grant P30-CA14051)National Institute of Environmental Health Sciences (Core Center Grant P30-ES002109)Singapore. Agency for Science, Technology and Research (National Science Scholarship)

In Vitro Culture, Drug Sensitivity, and Transcriptome of Plasmodium Vivax Hypnozoites

The unique relapsing nature of Plasmodium vivax infection is a major barrier to malaria eradication. Upon infection, dormant liver-stage forms, hypnozoites, linger for weeks to months and then relapse to cause recurrent blood-stage infection. Very little is known about hypnozoite biology; definitive biomarkers are lacking and in vitro platforms that support phenotypic studies are needed. Here, we recapitulate the entire liver stage of P. vivax in vitro, using a multiwell format that incorporates micropatterned primary human hepatocyte co-cultures (MPCCs). MPCCs feature key aspects of P. vivax biology, including establishment of persistent small forms and growing schizonts, merosome release, and subsequent infection of reticulocytes. We find that the small forms exhibit previously described hallmarks of hypnozoites, and we pilot MPCCs as a tool for testing candidate anti-hypnozoite drugs. Finally, we employ a hybrid capture strategy and RNA sequencing to describe the hypnozoite transcriptome and gain insight into its biology. Plasmodium vivax hypnozoites are difficult to study due to the lack of human liver platforms. Gural et al. recapitulated the entire liver stage of P. vivax in vitro, including formation and reactivation of hypnozoites and release of merosomes. Hybrid capture followed by RNA-seq revealed a first look into the hypnozoite transcriptome. Keywords: malaria; plasmodium vivax; liver stage; hypnozoite; RNA-seq; transcriptome; RNA capture; micropatterning; primary human hepatocytes; radical cureBill & Melinda Gates Foundation (Grant OPP1023607)National Cancer Institute (U.S.) (Grant P30-CA14051

Open Source Drug Discovery with the Malaria Box Compound Collection for Neglected Diseases and Beyond.

A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts

On Cartan matrices with two parameters (Cohomology theory of finite groups and related topics)

A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts

Antiprotozoal Malaria Box compounds with activity in biological assays and lacking toxicity at therapeutic levels.

<p>Selectivity Index, SI, is toxicity level/activity level; p, probe-like; d, drug-like.</p

Metabolomic and chemogenomic profiling.

<p>(A) Metabolic profiling: Heat map showing metabolic fingerprints of 80 Malaria Box compounds and atovaquone control. Parasite extracts were analyzed by LC-MS, and changes in metabolite pools were calculated for drug-treated parasites as compared to untreated controls. Hierarchical clustering was performed on ²log-fold changes in metabolites (data in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005763#ppat.1005763.s003" target="_blank">S2 Table</a>), scaled from -3 to +3. Six of seven compounds (indicated in red) reported to target <i>Pf</i>ATP4 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005763#ppat.1005763.ref025" target="_blank">25</a>] showed a distinct metabolic response characterized by the accumulation of dNTPs and a decrease in hemoglobin-derived peptides. A large cluster of compounds (indicated in blue) clustered with the atovaquone control (indicated in orange), and exhibit an atovaquone-like signature characterized by dysregulation of pyrimidine biosynthesis, and showed a distinct metabolic response characterized by the accumulation of dNTPs and a decrease in hemoglobin-derived peptides. (B) Chemogenomic profiling: A collection of 35 <i>P</i>. <i>falciparum</i> single insertion <i>piggyBac</i> mutants were profiled with 53 MMV compounds and 3 artemisinin (ART) compounds [Artesunate (AS), Artelinic acid (AL) and Artemether (AM)] for changes in IC₅₀ relative to the wild-type parent NF54 (data in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005763#ppat.1005763.s004" target="_blank">S3 Table</a>, genes queried in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005763#ppat.1005763.s005" target="_blank">S4 Table</a>). Clone PB58 carried a <i>piggyBac</i> insertion in the promoter region of the K13 gene and has an increased sensitivity to ART compounds as do PB54 and PB55 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005763#ppat.1005763.ref033" target="_blank">33</a>]. Drug-drug relationships based on similarities in IC₅₀ deviations of compounds generated with <i>piggyBac</i> mutants created chemogenomic profiles used to define drug-drug relationships. The significance of similarity in MoA between Malaria Box compounds and ART was evaluated by Pearson’s correlation calculations from pairwise comparisons. The X axis shows the chemogenomic profile correlation between a Malaria Box compound and AS, the Y axis with AM; the color gradient indicates the average correlation with all ART derivatives tested. Five Malaria Box compounds (MMV006087, MMV006427, MMV020492, MMV665876, MMV396797) were identified as having similar drug-drug chemogenomic profiles to the ART sensitivity cluster.</p