Pre-erythrocytic activity of M5717 in monotherapy and combination in preclinical Plasmodium infection models

Copyright © 2022 The Authors. Published by American Chemical SocietyCombination therapies have emerged to mitigate Plasmodium drug resistance, which has hampered the fight against malaria. M5717 is a potent multistage antiplasmodial drug under clinical development, which inhibits parasite protein synthesis. The combination of M5717 with pyronaridine, an inhibitor of hemozoin formation, displays potent activity against blood stage Plasmodium infection. However, the impact of this therapy on liver infection by Plasmodium remains unknown. Here, we employed a recently described 3D culture-based hepatic infection platform to evaluate the activity of the M5717-pyronaridine combination against hepatic infection by P. berghei. This effect was further confirmed in vivo by employing the C57BL/6J rodent Plasmodium infection model. Collectively, our data demonstrate that pyronaridine potentiates the activity of M5717 against P. berghei hepatic development. These preclinical results contribute to the validation of pyronaridine as a suitable partner drug for M5717, supporting the clinical evaluation of this novel antiplasmodial combination therapy.This work was funded by the healthcare business of Merck KGaA, Darmstadt, Germany (CrossRef Funder ID: 10.13039/100009945). M.P. is a recipient of a “Concurso de Estímulo ao Emprego Científico” Principal Investigator award of Fundação para a Ciência e Tecnologia, Portugal (FCT), with ref. N. CEECIND/03539/2017. D.F. is funded by FCT project CRCNA/BRB/0281/2019. F.A. is recipient of a PhD fellowship PD/BD/128371/2017, funded by FCT.info:eu-repo/semantics/publishedVersio

Refining iPSC-based 3D neural cell models and characterization tools to address brain microenvironment-related diseases

Brain microenvironment plays important roles in neurodevelopment and pathology and can affect therapy efficacy. Neural cell culture typically relies on the use of heterologous matrices that poorly resemble brain extracellular matrix (ECM) or reflect its pathological features. We have shown that perfusion bioreactor-based 3D differentiation of iPSC-derived human neural stem cells (hiPSC-NSC) sustains the concomitant differentiation of the three neural lineages (neurospheroids). If this neurospheroid culture strategy also allows deposition of native neural ECM it would be possible to (i) mimic cellular and microenvironment remodeling during neural differentiation, without the confounding effects of exogenous matrices and (ii) recapitulate pathological phenotypic features of diseases in which homotypic/ heterotypic cell-cell interactions and ECM are relevant. To characterize the neural extracellular space we employed quantitative transcriptomic (NGS) and proteome (SWATH-MS) analysis. Neurogenic developmental pathways were recapitulated in neurospheroids, with significant changes in cell membrane and ECM composition along differentiation; a significant enrichment in structural proteoglycans, typical of brain ECM, a downregulation of basement membrane proteins constituents and a higher expression of synaptic and ion transport machinery were observed. Neurospheroids were generated using hiPSC-NSC derived from Mucopolysaccharidosis type VII (MPS VII) patients. MPS VII is a rare neuronopathic lysosomal storage disease caused by deficient β-glucuronidase (β-gluc) activity, leading to glycosaminoglycan (GAGs) accumulation in the brain. The main MPS VII molecular hallmarks were recapitulated, e.g. accumulation og GAGs. By combining the neurospheroid culture with a 3D neuronal connectivity assay based on calcium imaging analysis we refined a new analytical strategy to characterize neuronal connectivity defects in a more predictive setting. We showed that MPS VII neurospheroids presented reduced neuronal activity and disturbances in network functionality, with alterations in connectivity and synchronization. These data provide insights into the interplay between reduced β-gluc activity, GAGs accumulation, alterations in neuronal network and its impact on MPS VII-associated cognitive defects Applying the characterization tools refined in this work to cope with 3D neurospheroid cultures, namely the neuronal connectivity assay, we provide a new platform to unveil the cellular processes responsible for brain dysfunction in neurological disorders and to test and optimize new therapies. Acknowledgments: iNOVA4Health – UID/Multi/04462/2013, a program financially supported by Fundação para a Ciência e Tecnologia/Ministério da Educação e Ciência, through national funds and co-funded by FEDER under the PT2020 Partnership Agreement, is acknowledged. PD/BD/52473/2014, PD/BD/52481/2014, and PD/BD/128371/2017 PhD fellowships funded by FCT, Portugal. N.R. is supported by the European Research Council Starting Grant 337327. MS data were obtained by UniMS – Mass Spectrometry Unit, ITQB and iBET, Oeiras, Portugal

Translation of liver stage activity of M5717, a Plasmodium elongation factor 2 inhibitor: from bench to bedside

© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.Background: Targeting the asymptomatic liver stage of Plasmodium infection through chemoprevention could become a key intervention to reduce malaria-associated incidence and mortality. Methods: M5717, a Plasmodium elongation factor 2 inhibitor, was assessed in vitro and in vivo with readily accessible Plasmodium berghei parasites. In an animal refinement, reduction, replacement approach, the in vitro IC99 value was used to feed a Population Pharmacokinetics modelling and simulation approach to determine meaningful effective doses for a subsequent Plasmodium sporozoite-induced volunteer infection study. Results: Doses of 100 and 200 mg would provide exposures exceeding IC99 in 96 and 100% of the simulated population, respectively. Conclusions: This approach has the potential to accelerate the search for new anti-malarials, to reduce the number of healthy volunteers needed in a clinical study and decrease and refine the animal use in the preclinical phase.This work was funded by the healthcare business of Merck KGaA, Darmstadt, Germany (CrossRef Funder ID: https://doi.org/10.13039/100009945). F.A. is the recipient of an individual Ph.D. fellowship funded by FCT (PD/BD/128371/2017).info:eu-repo/semantics/publishedVersio

iPSC-derived neurospheroids recapitulate development and pathological signatures of human brain microenvironment

Brain microenvironment plays an important role in neurodevelopment and pathology, where extracellular matrix (ECM) and soluble factors modulate multiple cellular processes. Neural cell culture typically relies on the use of heterologous matrices that poorly resemble the brain ECM or reflect its pathological features. We have previously demonstrated that perfusion stirred-tank bioreactor-based 3D differentiation of human neural stem cells (NSC) - pSTR-neurospheroids, sustains the concomitant differentiation of the three neural cell lineages (neurons, astrocytes and oligodendrocytes) and the establishment of physiologically relevant cell-cell interactions. Here, we hypothesized that if the pSTR-neurospheroid strategy would also allow the deposition of native neural ECM components and diffusion of secreted factors, it would be possible to: (i) mimic the cellular and microenvironment remodeling occurring during neural differentiation without the confounding effects of exogenous matrices; (ii) recapitulate the pathological phenotypes of diseases in which alteration of homotypic and heterotypic cell-cell interactions and ECM components are relevant. To demonstrate the first point, we analyzed pSTR-neurospheroid differentiation by quantitative transcriptome (NGS) and proteome (SWATH-MS). Data showed that neurogenic developmental pathways were recapitulated, with significant changes at cell membrane and ECM composition, diverging from the 2D differentiation profile. A significant enrichment in structural proteoglycans typical of brain ECM, along with downregulation of basement membrane constituents was observed. Moreover, higher expression of synaptic and ion transport machinery in pSTR-neurospheroids suggest higher neuronal maturation than in 2D. Having shown recapitulation of neural microenvironmental dynamics in pSTR-neurospheroids, we used Mucopolysaccharidosis VII (MPSVII) as a disease case study. MPS VII is a lysosomal storage disease caused by deficient β-glucuronidase (β-gluc) activity, which leads to accumulation of glycosaminoglycans (GAGs) in many tissues, including the brain. In pSTR-neurospheroids generated from hiPSC of a MPS VII patient, the main molecular disease hallmarks were recapitulated, namely accumulation of GAGs. Notably, MPS VII neurospheroids showed reduced neuronal activity and a disturbance in network functionality, with alterations both in connectivity and synchronization, not observed in 2D cultures. These data provide insight into the interplay between reduced β-gluc activity, GAG accumulation, alterations in the neural network, and its impact on MPS VII-associated cognitive defects. Overall we demonstrate that neural cellular and extracellular developmental and pathological features are recapitulated in healthy and diseased pSTR-neurospheroids, respectively. These can be valuable in vitro models to address molecular defects associated with neurological disorders that affect neural microenvironment homeostasis. Moreover, the 3D neuronal connectivity assay developed is a new tool with potential to assess other lysosomal storage diseases and neurodegenerative diseases that have variable phenotypes. Acknowledgements: SFRH/BD/78308/2011, SFRH/BD/52202/2013 and SFRH/BD/52473/2014 PhD fellowships from FCT, Portugal and iNOVA4Health-UID/Multi/04462/2013, supported by FCT/ MEC, through national funds and co-funded by FEDER under the PT2020 Partnership Agreement

Advancing liver stage malaria in vitro models for drug discovery

"The malaria drug pipeline has been highly affected by the fastdeveloping rate of Plasmodium resistance to the currently available drugs. There are two promising and complementary approaches being explored to mitigate drug resistance. One is focused on the drugs, and includes the development of drugs exploiting novel targets and the combination of drug partners presenting the same pharmacokinetic properties but distinct modes of action. The other approach is to explore the life cycle of the parasite and target the hepatic stage of the Plasmodium life cycle.(...)"Merck KGaA Darmstadt, German

Bioengineered Liver Cell Models of Hepatotropic Infections

Hepatitis viruses and liver-stage malaria are within the liver infections causing higher morbidity and mortality rates worldwide. The highly restricted tropism of the major human hepatotropic pathogens—namely, the human hepatitis B and C viruses and the Plasmodium falciparum and Plasmodium vivax parasites—has hampered the development of disease models. These models are crucial for uncovering the molecular mechanisms underlying the biology of infection and governing host–pathogen interaction, as well as for fostering drug development. Bioengineered cell models better recapitulate the human liver microenvironment and extend hepatocyte viability and phenotype in vitro, when compared with conventional two-dimensional cell models. In this article, we review the bioengineering tools employed in the development of hepatic cell models for studying infection, with an emphasis on 3D cell culture strategies, and discuss how those tools contributed to the level of recapitulation attained in the different model layouts. Examples of host–pathogen interactions uncovered by engineered liver models and their usefulness in drug development are also presented. Finally, we address the current bottlenecks, trends, and prospect toward cell models’ reliability, robustness, and reproducibility

Recapitulation of Human Neural Microenvironment Signatures in iPSC-Derived NPC 3D Differentiation

Brain microenvironment plays an important role in neurodevelopment and pathology, where the extracellular matrix (ECM) and soluble factors modulate multiple cellular processes. Neural cell culture typically relies on heterologous matrices poorly resembling brain ECM. Here, we employed neurospheroids to address microenvironment remodeling during neural differentiation of human stem cells, without the confounding effects of exogenous matrices. Proteome and transcriptome dynamics revealed significant changes at cell membrane and ECM during 3D differentiation, diverging significantly from the 2D differentiation. Structural proteoglycans typical of brain ECM were enriched during 3D differentiation, in contrast to basement membrane constituents in 2D. Moreover, higher expression of synaptic and ion transport machinery was observed in 3D cultures, suggesting higher neuronal maturation in neurospheroids. This work demonstrates that 3D neural differentiation as neurospheroids promotes the expression of cellular and extracellular features found in neural tissue, highlighting its value to address molecular defects in cell-ECM interactions associated with neurological disorders. In this article, Brito and colleagues show that differentiation of human neural stem cells as three-dimensional neurospheroids induces significant changes at cell membrane and ECM composition, relative to monolayer cultures, mimicking a neural-like microenvironment. These findings support the potential of neurospheroids to address ECM-affecting neurological disorders.publishersversionpublishe

Flexible 3D cell-based platforms for the discovery and profiling of novel drugs targeting plasmodium hepatic infection

The restricted pipeline of drugs targeting the liver stage of; Plasmodium; infection reflects the scarcity of cell models that mimic the human hepatic phenotype and drug metabolism, as well as; Plasmodium; hepatic infection. Using stirred-tank culture systems, spheroids of human hepatic cell lines were generated, sustaining a stable hepatic phenotype over 4 weeks of culture. Spheroids were employed in the establishment of 3D; Plasmodium berghei; infection platforms that relied on static or dynamic culture conditions.; P. berghei; invasion and development were recapitulated in the hepatic spheroids, yielding blood-infective merozoites. The translational potential of the 3D platforms was demonstrated by comparing the; in vitro; minimum inhibitory concentration of M5717, a compound under clinical development, with; in vivo; plasma concentrations that clear liver stage; P. berghei; in mice. Our results show that the 3D platforms are flexible and scalable and can predict the efficacy of antiplasmodial therapies, constituting a powerful tool for integration in drug discovery programs