Biology subjects

Sakura, T.

Publications and source records attributed to Sakura, T..

9 recordsLinked to original sources

bioRxiv2026

Structural insights and mechanistic inhibition of Plasmodium falciparum dihydroorotate dehydrogenase by newly identified chemotypes

Resistance to frontline antimalarial drugs continues to threaten malaria control, highlighting the need for new chemotypes. Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) catalyzes a key step in de novo pyrimidine biosynthesis and remains a validated antimalarial target. Although the PfDHODH inhibitor DSM265 advanced to clinical development, resistance-associated mutations highlight the need for structurally distinct inhibitors. Here, we report the discovery and structural characterization of PfDHODH inhibitors identified through target-based high-throughput screening of 432,592 compounds. Primary hits were evaluated by dose-response assays against asexual blood-stage parasites, mammalian mitochondrial toxicity assays, and HepG2 cytotoxicity profiling, yielding 348 hits. Seven representative compounds were prioritized based on efficacy against PfDHODH and orthologs from P. vivax and P. berghei, and their X-ray co-crystal structures revealed distinct binding modes within the active site. Ferulenol exhibited sub-micromolar inhibition of recombinant PfDHODH (rPfDHODH) and rPvDHODH, together with potent blood-stage activity, with its prenylated tail contributing additional hydrophobic interactions. Compound 2 showed the highest potency across the tested DHODH enzymes but reduced selectivity for PfDHODH, potentially due to its distinct binding orientation and furylmethyl substituent. Comparative structural analyses with DSM265 identified binding-site features that may guide the design of next-generation PfDHODH inhibitors with improved potency and selectivity.

microbiology↗

bioRxiv2025

Plasmodium falciparum diacylglycerol acyltransferase maintains phospholipid homeostasis to regulate sexual differentiation, ER stress, and cytoadhesion

Plasmodium falciparum is the causative agent of human malaria, a life-threating infectious disease that imposes a major global health burden. Lipid metabolism is indispensable for this parasites replication and survival, yet most of the molecular components and mechanisms involved remain poorly understood. In eukaryotes, lipid droplets (LDs) serve as dynamic organelles that store neutral lipids (NLs), buffer lipotoxic stress, and regulate signaling pathways, with their biogenesis controlled by diacylglycerol o-acyltransferases (DGATs). Although P. falciparum encodes a putative DGAT (PF3D7_0322300), its role in the parasite life cycle has not been elucidated. We generated conditional PfDGAT-knockout parasites to investigate the enzymes functional significance. PfDGAT deficiency led to parasite death, accompanied by reduced LD formation, elevated phospholipid levels, and induction of ER stress. Moreover, PfDGAT deletion altered protein trafficking, resulting in the decreased cytoadherence of parasite-infected erythrocytes to human brain microvascular endothelial cells, and suppressed parasite sexual differentiation. Thus, PfDGAT deletion affected multiple aspects of the parasites life cycle, highlighting its critical role in parasite survival and pathogenesis. Our findings provide new insights into parasite lipid homeostasis and highlight DGAT as a potential target of antimalarial intervention.

microbiology↗

bioRxiv2025

Efficient identification of antimalarials with novel mechanism of action via multi-sample transcriptomic profiling

Malaria poses a significant health burden to endemic countries, necessitating the development of new antimalarials with novel mechanisms of action to combat emerging drug resistance. However, target identification methods for antimalarial compounds selected through phenotypic screening are currently limited, which has been a major obstacle in the field of antimalarial drug discovery. Here, we aim to address this need using perturbation transcriptomics to efficiently select antimalarial compounds targeting new pathways or molecules. The method involves comparing the transcriptomic changes by drug perturbation in the asexual blood stage of the malaria parasite Plasmodium falciparum. Using this method, we identified several compounds that exhibited distinct transcriptomic profiling from a diverse set of antimalarials with known modes of action, indicating their potential to target different pathways. In addition, we successfully identified a novel histone deacetylase inhibitor using this approach and chemogenetically validated its target through in vitro evolution. These results highlight the effectiveness of this platform in accelerating antimalarial drug discovery.

microbiology↗

bioRxiv2025

Epigenetic landscape of the H3K27me3 mark in macrophages transformed by Theileria annulata

Theileria annulata and T. parva are obligate intracellular parasites that induce bovine leukocyte transformation leading to uncontrolled proliferation and heightened dissemination of infected leukocytes. Early passage T. annulata-transformed macrophages are virulent, but with long-term culture, they become attenuated for dissemination. Loss of dissemination is restored when TGF-{beta}2-mediated signaling is re-established in attenuated macrophages, suggesting that epigenetic changes might be occurring that contribute to the attenuated phenotype. We focused on one of the important repressive histone marks, tri-methylated lysine 27 of histone H3 (H3K27me3) catalysed by the Polycomb Repressive Complex 2 and found that the global level of H3K27me3 is increased in attenuated macrophages. ChIP-seq revealed the genomic distribution of H3K27me3 is heavily remodeled from virulent to attenuated macrophages, where many genes transition from a focal peak of H3K27me3 around the transcription start site to larger chromatin domains. This transition is not paralleled by large-scale transcriptional downregulation. RNA-seq analysis following PRC2 inhibitor treatment reveals that fewer genes are derepressed in attenuated macrophages than in virulent macrophages, suggesting that broader H3K27me3 profiles do not systematically translate into increased gene silencing activity. Our findings shed light on the mechanisms underlying the dysregulation of epigenetic modifications in Theileria-induced leukocyte transformation. Significance statementTropical theileriosis is a major constraint to livestock production worldwide. Infection with Theileria annulata induces the transformation of bovine leukocytes, which subsequently exhibit similarities to human leukemia. Long-term in vitro culture of transformed leukocytes attenuates their tumorigenic properties. The molecular changes underlying this process are currently unclear. We focused on H3K27me3, a key histone tail post-translational modification that regulates chromatin structure and gene expression, and compared its genomic landscapes in virulent, highly disseminating macrophages versus attenuated macrophages with reduced dissemination potential that are used as a live vaccine against tropical theileriosis. Western blot and ChIP-seq revealed higher global levels of H3K27me3 and a striking broadening of its genomic distribution in attenuated macrophages. To our surprise, this reconfiguration is not associated with major changes in gene expression and attenuated cells appear to be less responsive to PRC2 inhibition, suggesting that large-scale broadening of H3K27me3 does not systematically translate into increased PRC2-mediated silencing activity. Despite these changes, H3K27me3 maintains the silencing of key tumor suppressor genes in both virulent and attenuated macrophages. These findings shed light on the interplay between infection, the evolution of oncogenic potential and reconfiguration of the chromatin landscape following attenuation of virulence.

microbiology↗

bioRxiv2024

Accelerating antimalarial drug discovery with a new high-throughput screen for fast-killing compounds

The urgent need for rapidly acting compounds in the development of antimalarial drugs underscores the significance of such compounds in overcoming resistance issues and improving patient adherence to antimalarial treatments. The present study introduces a high-throughput screening (HTS) approach using 1536-well plates, employing Plasmodium falciparum lactate dehydrogenase (PfLDH) combined with nitroreductase (NTR) and fluorescent probes to evaluate inhibition of the growth of the asexual blood stage of malaria parasites. This method was adapted to efficiently measure the parasite reduction ratio (PRR) in a 384-well plate format, streamlining the traditionally time-consuming screening process. By successfully screening numerous compounds, this approach identified fast-killing hits early in the screening process, addressing challenges associated with artemisinin-based combination therapies. The high-throughput PRR method is expected to be of value in continuously monitoring fast-killing properties during structure-activity relationship studies, expediting the identification and development of novel, rapidly acting antimalarial drugs within phenotypic drug discovery campaigns.

microbiology↗

bioRxiv2024

Glycolytic enzymes form membrane-less condensates in the malaria parasite Plasmodium falciparum by sensing glucose levels

Recent studies have shown that liquid-liquid phase separation (LLPS) in cells can regulate essential cellular events, including metabolic processes. Glycolytic bodies (G-bodies) are biomolecular condensates formed through the LLPS of glycolytic enzymes, and they accelerate glycolysis to overcome hypoxic stress in several organisms. Although the asexual blood stage (ABS) of the human malaria parasite Plasmodium falciparum highly depends on glycolysis for energy production, there have been no reports of the formation of such G-bodies throughout the parasites lifecycle. Using fluorescence tagging and live imaging, we found that G-body-like condensates containing phosphofructokinase 9 (PFK9) and phosphoglycerate kinase (PGK) were formed in the parasite cells after long-term culture under conditions of low glucose. These G-body-like structures appeared stable, but membrane staining and osmotic stress experiments suggested that the observed condensates were not associated with lipid membrane. Further microscopic observations and mathematical analyses of high signal-to-noise ratio images indicated that small condensates were formed transiently first, and these then gradually grew and stabilized in the cytosol. These results suggested that the formation of glycolytic enzyme condensates may be an important cellular response for adapting to blood sugar level oscillations in the host and maintaining the parasites multiplication in the ABS. Significance statementGlycolytic bodies (G-bodies), which are biomolecular condensates formed through the liquid-liquid phase separation of glycolytic enzymes, can accelerate glycolysis to produce energy and overcome hypoxic stress. The parasites that cause malaria depend on glycolysis for energy production, but there have been no reports that these parasites form G-bodies. We demonstrated that membrane-less G-body-like structures formed in media containing low levels of glucose. Small condensates appeared first and over time, the condensates became larger and more stable. The formation of glycolytic enzyme condensates may be important for the malaria parasite to adapt to fluctuating blood sugar levels in the host. These results further our understanding of the cellular mechanisms for the survival of malaria parasites.

cell biology↗

bioRxiv2024

Pivotal roles of Plasmodium falciparum lysophospholipid acyltransferase 1 in cell cycle progression and cytostome internalization

The rapid intraerythrocytic replication of Plasmodium falciparum, a deadly species of malaria parasite, requires a quick but constant supply of phospholipids to support marked cell membrane expansion. In the malarial parasite, many enzymes functioning in phospholipid synthesis pathway have not been identified or characterized. Here, we identified P. falciparum lysophospholipid acyltransferase 1 (PfLPLAT1) and showed that PfLPLAT1 is vital for asexual parasite cell cycle progression and cytostome internalization. Deficiency in PfLPLAT1 resulted in decreased parasitemia and prevented transition to the schizont stage. Parasites lacking PfLPLAT1 also exhibited distinctive omega-shaped vacuoles, indicating disrupted cytostome function. Transcriptomic analyses suggested that this deficiency impacted DNA replication and cell cycle regulation. Mass spectrometry-based enzyme assay and lipidomic analysis demonstrated that recombinant PfLPLAT1 exhibited lysophospholipid acyltransferase activity with a preference for unsaturated fatty acids as its acyl donors and lysophosphatidic acids as an acceptor, with its conditional knockout leading to abnormal lipid composition and marked morphological and developmental changes including stage arrest. These findings highlight PfLPLAT1 as a potential target for antimalarial therapy, particularly due to its unique role and divergence from human orthologs.

microbiology↗

bioRxiv2020

5-amino levulinic acid inhibits SARS-CoV-2 infection in vitro

The current COVID-19 pandemic requires urgent development of effective therapeutics. 5-amino levulinic acid (5-ALA) is a naturally synthesized amino acid and has been used for multiple purposes including as an anticancer therapy and as a dietary supplement due to its high bioavailability. In this study, we demonstrated that 5-ALA treatment potently inhibited infection of SARS-CoV-2, a causative agent of COVID-19. The antiviral effects could be detected in both human and non-human cells, without significant cytotoxicity. Therefore, 5-ALA is a candidate as an oral antiviral drug for COVID-19.

microbiology↗

bioRxiv2019

Evaluation of 3D markerless motion capture accuracy using OpenPose with multiple video cameras

There is a need within human movement sciences for a markerless motion capture system, which is easy to use and suffciently accurate to evaluate motor performance. This study aims to develop a 3D markerless motion capture technique, using OpenPose with multiple synchronized video cameras, and examine its accuracy in comparison with optical marker-based motion capture. Participants performed three motor tasks (walking, countermovement jumping, and ball throwing), with these movements measured using both marker-based optical motion capture and OpenPose-based markerless motion capture. The differences in corresponding joint positions, estimated from the two different methods throughout the analysis, were presented as a mean absolute error (MAE). The results demonstrated that, qualitatively, 3D pose estimation using markerless motion capture could correctly reproduce the movements of participants. Quantitatively, of all the mean absolute errors calculated, approximately 47% were less than 20 mm and 80% were less than 30 mm. However, 10% were greater than 40 mm. The primary reason for mean absolute errors exceeding 40mm was that OpenPose failed to track the participants pose in 2D images owing to failures, such as recognition of an object as a human body segment, or replacing one segment with another depending on the image of each frame. In conclusion, this study demonstrates that, if an algorithm that corrects all apparently wrong tracking can be incorporated into the system, OpenPose-based markerless motion capture can be used for human movement science with an accuracy of 30mm or less.

neuroscience↗