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The Plasmodium falciparum proteasome is a potential antimalarial drug target. We have identified a series of amino-amide boronates that are potent and specific inhibitors of the P. falciparum 20S proteasome (Pf20S) beta5 active site and that exhibit fast-acting antimalarial activity. They selectively inhibit the growth of P. falciparum compared with a human cell line and exhibit high potency against field isolates of P. falciparum and Plasmodium vivax They have a low propensity for development of resistance and possess liver stage and transmission-blocking activity. Exemplar compounds, MPI-5 and MPI-13, show potent activity against P. falciparum infections in a SCID mouse model with an oral dosing regimen that is well tolerated. We show that MPI-5 binds more strongly to Pf20S than to human constitutive 20S (Hs20Sc). Comparison of the cryo-electron microscopy (EM) structures of Pf20S and Hs20Sc in complex with MPI-5 and Pf20S in complex with the clinically used anti-cancer agent, bortezomib, reveal differences in binding modes that help to explain the selectivity. Together, this work provides insights into the 20S proteasome in P. falciparum, underpinning the design of potent and selective antimalarial proteasome inhibitors.

© 2017 Hopp et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited., Proteases have been implicated in a variety of developmental processes during the malaria parasite lifecycle. In particular, invasion and egress of the parasite from the infected hepatocyte and erythrocyte, critically depend on protease activity. Although falcipain-1 was the first cysteine protease to be characterized in P. falciparum, its role in the lifecycle of the parasite has been the subject of some controversy. While an inhibitor of falcipain-1 blocked erythrocyte invasion by merozoites, two independent studies showed that falcipain-1 disruption did not affect growth of blood stage parasites. To shed light on the role of this protease over the entire Plasmodium lifecycle, we disrupted berghepain-1, its ortholog in the rodent parasite P. berghei. We found that this mutant parasite displays a pronounced delay in blood stage infection after inoculation of sporozoites. Experiments designed to pinpoint the defect of berghepain-1 knockout parasites found that it was not due to alterations in gliding motility, hepatocyte invasion or liver stage development and that injection of berghepain-1 knockout merosomes replicated the phenotype of delayed blood stage growth after sporozoite inoculation. We identified an additional role for berghepain-1 in preparing blood stage merozoites for infection of erythrocytes and observed that berghepain-1 knockout parasites exhibit a reticulocyte restriction, suggesting that berghepain-1 activity broadens the erythrocyte repertoire of the parasite. The lack of berghepain-1 expression resulted in a greater reduction in erythrocyte infectivity in hepatocyte-derived merozoites than it did in erythrocyte-derived merozoites. These observations indicate a role for berghepain-1 in processing ligands important for merozoite infectivity and provide evidence supporting the notion that hepatic and erythrocytic merozoites, though structurally similar, are not identical., This work was supported by the National Institutes of Health (R01 grant A1056840 to PS), a Johns Hopkins Malaria Research Institute fellowship (CSH) (malaria.jhsph.edu/opportunities/fellowships/) and the Netherlands Organization for Scientific Research Rubicon fellowship (WAvdL) (www.nwo.nl/funding-rubicon).

Summary Organisms have evolved numerous strategies to control infection by an array of intracellular pathogens. One cell autonomous pathogen control strategy is global inhibition of protein synthesis via stress granule (SG) formation. SGs are induced by stressful stimuli such as oxidative stress and nutrient deprivation, and are known to counteract both viral and bacterial infections. Pathogens, in turn, may actively block an infected cell's ability to form SGs. In vitro and in vivo, many liver stage malaria parasites are eliminated during development. We show here that SG formation is not amongst the strategies used for elimination of parasites from hepatocytes. Neither cell traversal, sporozoite invasion, nor rapid parasite growth leads to the formation of SGs. Furthermore, Plasmodium berghei infection does not compromise the ability of infected cells to assemble SGs in response to oxidative or nutritional stress. Plasmodium infection is therefore not detected by hepatocytes as a strong stressor necessitating global translational repression in response, highlighting the idea that Plasmodium has evolved strategies to ensure its remarkable growth in the hepatocyte while maintaining host cell homeostasis.

Before they infect red blood cells and cause malaria, Plasmodium parasites undergo an obligate and clinically silent expansion phase in the liver that is supposedly undetected by the host. Here, we demonstrate the engagement of a type I interferon (IFN) response during Plasmodium replication in the liver. We identified Plasmodium RNA as a previously unrecognized pathogen-associated molecular pattern (PAMP) capable of activating a type I IFN response via the cytosolic pattern recognition receptor Mda5. This response, initiated by liver-resident cells through the adaptor molecule for cytosolic RNA sensors, Mavs, and the transcription factors Irf3 and Irf7, is propagated by hepatocytes in an interferon-α/β receptor-dependent manner. This signaling pathway is critical for immune cell-mediated host resistance to liver-stage Plasmodium infection, which we find can be primed with other PAMPs, including hepatitis C virus RNA. Together, our results show that the liver has sensor mechanisms for Plasmodium that mediate a functional antiparasite response driven by type I IFN.

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., Author Summary Malaria leads to the loss of over 440,000 lives annually; accelerating research to discover new candidate drugs is a priority. Medicines for Malaria Venture (MMV) has distilled over 25,000 compounds that kill malaria parasites in vitro into a group of 400 representative compounds, called the "Malaria Box". These Malaria Box sets were distributed free-of-charge to research laboratories in 30 different countries that work on a wide variety of pathogens. Fifty-five groups compiled >290 assay results for this paper describing the many activities of the Malaria Box compounds. The collective results suggest a potential mechanism of action for over 130 compounds against malaria and illuminate the most promising compounds for further malaria drug development research. Excitingly some of these compounds also showed outstanding activity against other disease agents including fungi, bacteria, other single-cellular parasites, worms, and even human cancer cells. The results have ignited over 30 drug development programs for a variety of diseases. This open access effort was so successful that MMV has begun to distribute another set of compounds with initial activity against a wider range of infectious agents that are of public health concern, called the Pathogen Box, available now to scientific labs all over the world (www.PathogenBox.org).

Copyright © 2015, American Society for Microbiology. All Rights Reserved., Prior to invading nonreplicative erythrocytes, Plasmodium parasites undergo their first obligate step in the mammalian host inside hepatocytes, where each sporozoite replicates to generate thousands of merozoites. While normally quiescent, hepatocytes retain proliferative capacity and can readily reenter the cell cycle in response to diverse stimuli. Many intracellular pathogens, including protozoan parasites, manipulate the cell cycle progression of their host cells for their own benefit, but it is not known whether the hepatocyte cell cycle plays a role during Plasmodium liver stage infection. Here, we show that Plasmodium parasites can be observed in mitotic hepatoma cells throughout liver stage development, where they initially reduce the likelihood of mitosis and ultimately lead to significant acquisition of a binucleate phenotype. However, hepatoma cells pharmacologically arrested in S phase still support robust and complete Plasmodium liver stage development, which thus does not require cell cycle progression in the infected cell in vitro. Furthermore, murine hepatocytes remain quiescent throughout in vivo infection with either Plasmodium berghei or Plasmodium yoelii, as do Plasmodium falciparum-infected primary human hepatocytes, demonstrating that the rapid and prodigious growth of liver stage parasites is accomplished independent of host hepatocyte cell cycle progression during natural infection., This work was supported by funding from the European Research Council under the European Union's 7th Framework Programme (FP7/2007-2013) through ERC grant agreement number 311502 (M.M.M.) and from Fundação para a Ciência e Tecnologia (FCT; Portugal) through grant number EXCL/IMI-MIC/0056/2012 (M.M.M.). K.K.H. was supported by funds from the European Community's Seventh Framework Programme (FP7/2007-2013) Marie Cure IEF (PIEF-GA-2008-221854) and Fundação para a Ciência e Tecnologia (SFRH/BPD/40989/2007).

Contains fulltext : 177492.pdf (Publisher’s version ) (Closed access) As part of the global effort toward malaria eradication, phenotypic whole-cell screening revealed the 2-aminopyridine class of small molecules as a good starting point to develop new antimalarial drugs. Stemming from this series, we found that the derivative, MMV390048, lacked cross-resistance with current drugs used to treat malaria. This compound was efficacious against all Plasmodium life cycle stages, apart from late hypnozoites in the liver. Efficacy was shown in the humanized Plasmodium falciparum mouse model, and modest reductions in mouse-to-mouse transmission were achieved in the Plasmodium berghei mouse model. Experiments in monkeys revealed the ability of MMV390048 to be used for full chemoprotection. Although MMV390048 was not able to eliminate liver hypnozoites, it delayed relapse in a Plasmodium cynomolgi monkey model. Both genomic and chemoproteomic studies identified a kinase of the Plasmodium parasite, phosphatidylinositol 4-kinase, as the molecular target of MMV390048. The ability of MMV390048 to block all life cycle stages of the malaria parasite suggests that this compound should be further developed and may contribute to malaria control and eradication as part of a single-dose combination treatment.

We identified Drosophila Smurf (DSmurf) as a negative regulator of signaling by the BMP2/4 ortholog DPP during embryonic dorsal-ventral patterning. DSmurf encodes a HECT domain ubiquitin-protein ligase, homologous to vertebrate Smurf1 and Smurf2, that binds the Smad1/5 ortholog MAD and likely promotes its proteolysis. The essential function of DSmurf is restricted to its action on the DPP pathway. DSmurf has two distinct, possibly mechanistically separate, functions in controlling DPP signaling. Prior to gastrulation, DSmurf mutations cause a spatial increase in the DPP gradient, as evidenced by ventrolateral expansion in expression domains of target genes representing all known signaling thresholds. After gastrulation, DSmurf mutations cause a temporal delay in downregulation of earlier DPP signals, resulting in a lethal defect in hindgut organogenesis.

Homeostasis of mammalian cell function strictly depends on balancing oxygen exposure to maintain energy metabolism without producing excessive reactive oxygen species. In vivo, cells in different tissues are exposed to a wide range of oxygen concentrations, and yet in vitro models almost exclusively expose cultured cells to higher, atmospheric oxygen levels. Existing models of liver stage malaria that utilize primary human hepatocytes typically exhibit low in vitro infection efficiencies, possibly due to missing microenvironmental support signals. One cue that may influence the infection capacity of cultured human hepatocytes is the dissolved oxygen concentration. We developed a microscale human liver platform comprised of precisely patterned primary human hepatocytes and nonparenchymal cells (MPCC) to model liver stage malaria, but the oxygen concentrations are typically higher in the in vitro liver platform than anywhere along the hepatic sinusoid. Indeed, we observed that liver stage Plasmodium parasite development in vivo correlates with hepatic sinusoidal oxygen gradients. Therefore, we hypothesized that in vitro liver stage malaria infection efficiencies may improve under hypoxia. Using the infection of MPCCs with P. berghei or P. yoelii as a model, we observed that ambient hypoxia resulted in increased survival of exo-erythrocytic forms (EEFs) in hepatocytes, and improved parasite development in a subset of surviving EEFs, based on EEF size. Further, the effective cell surface oxygen tensions (pO2) experienced by the hepatocytes, as predicted by a mathematical model, were systematically perturbed by varying culture parameters like hepatocyte density and media height, uncovering an optimal cell surface pO2 to maximize the number of mature EEFs. Initial mechanistic experiments reveal that treatment of primary human hepatocytes with the hypoxia mimetic, cobalt (II) chloride, as well as a HIF-1α activator, dimethyloxalylglycine, also enhance P. berghei infection, suggesting that the effect of hypoxia on infection is mediated in part by host-dependent HIF-1α mechanisms., Bill & Melinda Gates Foundation (Award 51066), Howard Hughes Medical Institute

This deposit is in restrictedAccess (it can't be in open access to the public), and can only be accessed by two ways: either by requesting a legal copy from the author (the email contact present in this deposit) or by visiting the following link: https://www.sciencedirect.com/science/article/pii/S0008874912001153?via%3Dihub Although the role of regulatory T cells (Tregs) during malaria infection has been studied extensively, such studies have focused exclusively on the role of Treg during the blood stage of infection; little is known about the detailed mechanisms of Tregs and sporozoite deposition in the dermis by mosquito bites. In this paper we show that sporozoites introduced into the skin by mosquito bites increase the mobility of skin Tregs and dendritic cells (DCs). We also show differences in MHC class II and/or CD86 expression on skin-resident dendritic cell subtypes and macrophages. From the observed decrease of the number of APCs into draining lymph nodes, suppression of CD28 expression in conventional CD4 T cells, and a low homeostatic proliferation of skin-migrated CD4 T found in nude mice indicate that Tregs may play a fundamental role during the initial phase of malaria parasite inoculation into the mammalian host

Copyright © 2013 National Academy of Sciences., Residence within a customized vacuole is a highly successful strategy used by diverse intracellular microorganisms. The parasitophorous vacuole membrane (PVM) is the critical interface between Plasmodium parasites and their possibly hostile, yet ultimately sustaining, host cell environment. We show that torins, developed as ATP-competitive mammalian target of rapamycin (mTOR) kinase inhibitors, are fast-acting antiplasmodial compounds that unexpectedly target the parasite directly, blocking the dynamic trafficking of the Plasmodium proteins exported protein 1 (EXP1) and upregulated in sporozoites 4 (UIS4) to the liver stage PVM and leading to efficient parasite elimination by the hepatocyte. Torin2 has single-digit, or lower, nanomolar potency in both liver and blood stages of infection in vitro and is likewise effective against both stages in vivo, with a single oral dose sufficient to clear liver stage infection. Parasite elimination and perturbed trafficking of liver stage PVM-resident proteins are both specific aspects of torin-mediated Plasmodium liver stage inhibition, indicating that torins have a distinct mode of action compared with currently used antimalarials., This work was supported by Fundação para a Ciência e Tecnologia (FCT, Portugal) Grants PTDC/SAU-GMG/100313/2008 and EXCL/IMI-MIC/0056/2012, and European Research Council funding (to M.M.M.). K.K.H. was supported by funds from the European Community’s Seventh Framework Programme (FP7/2007-2013) Marie Curie IntraEuropean Fellowship Grant PIEF-GA-2008-221854 and FCT Grant SFRH/BPD/40989/2007.