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Results for “high-throughput screening (HTS)”

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PubMed2026

Development of a cell-based nanoluciferase reporter system for high-throughput screening of HBV cccDNA inhibitors.

Hepatitis B virus (HBV) persistence is sustained by the viral covalently closed circular DNA (cccDNA) minichromosome, which remains a major barrier to curative antiviral therapies. The lack of reliable quantitative cccDNA detection methods and surrogate markers has hindered efforts to target cccDNA in antiviral high-throughput screening (HTS). Here, we established a novel inducible cccDNA-dependent nanoluciferase (NLuc) reporter cell line, designated HepBLE12, by inserting an in-frame 11-amino acid split-NLuc HiBiT tag into the precore (pC) coding region of an HBV transgene. The resulting 1.3-kDa HiBiT tag on pC serves as the detection module of the split NLuc system, generating quantitative luminescence upon high-affinity complementation with the cognate 18-kDa LgBiT subunit in cell lysates. Notably, the HiBiT assay enables direct detection of intracellular HiBiT-pC protein rather than secreted HBeAg, providing a reporter signal more closely linked to cccDNA activity. HepBLE12 cells exhibited inducible and robust viral DNA replication, and the cccDNA-dependent HiBiT signal was validated under diverse experimental conditions that modulate cccDNA formation or transcription. We further miniaturized the assay to a 384-well format and optimized key parameters following standard HTS assay development practices. The assay was successfully automated and demonstrated excellent performance in a multi-day variability study and a pilot screen, with signal-to-background (S/B) ≈ 9, coefficient of variance (CV) < 10%, and average Z-factor value of 0.74, exceeding canonical HTS quality benchmarks. Together, the HepBLE12 cell-based HTS platform provides a robust and practical tool for identifying inhibitors targeting HBV cccDNA.

Hepatitis B virus↗

PubMed2026

Genetic and biochemical screens identify MGAT1 as a druggable glycosyltransferase target in STK11-mutant lung cancer.

Checkpoint inhibitors are standard-of-care therapies for non-small cell lung cancer (NSCLC), but their efficacy is limited in tumors with STK11 mutations, highlighting the need for new therapeutic strategies. Here, we performed complementary in vivo and in vitro CRISPR-Cas9 functional genomic screens to identify genes whose loss restores sensitivity to anti-PD-1 therapy. We found that loss of MGAT1, a Golgi glycosyltransferase critical for the maturation of high-mannose N-glycans into hybrid and complex glycan structures, reversed resistance to anti-PD-1 treatment in syngeneic mouse tumor models harboring STK11 mutations. Parallel co-culture screens with antigen-matched CD8+ T cells further showed that disruption of N-glycosylation strongly sensitized tumor cells to T cell-mediated killing. Genetic rescue studies demonstrated that this immune-evasion phenotype depends on MGAT1 catalytic activity, supporting direct biochemical interrogation of the enzyme. Using purified human MGAT1 and a UDP-Glo™ glycosyltransferase assay, we established a tractable screening platform and performed a 500,000-compound biochemical high-throughput screen, identifying an initial hit (compound 1; IC50 = 197 μM). Subsequent medicinal chemistry optimization delivered progressively more potent analogs, including TNG-9333 (0.814 μM) and TNG-2673 (0.043 μM) and represented a >1000-fold improvement in biochemical potency from the starting hit. Crystal structures of human MGAT1 in apo, UDP-bound, UDP-GlcNAc-bound, and inhibitor-bound states, together with SPR and DSF analyses, revealed that this chemical series engages a previously unrecognized allosteric pocket and inhibits MGAT1 through a UDP-noncompetitive mechanism. Collectively, our work implicates N-glycosylation as a key mediator of immune evasion and establishes MGAT1 as a ligandable, structurally tractable target for small-molecule drug discovery.

CRISPR/Cas9 target discovery↗

PubMed2025

Characterization of a PRKCE::ETV6 fusion as a potential oncogenic driver in T-cell acute lymphoblastic leukemia.

BACKGROUND: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy caused by mutation accumulation during hematopoiesis. The characterization of chromosomal abnormalities may provide significant insights into genetic mechanisms of malignant transformation in hematopoietic cells. However, T-ALL is genetically very heterogenous and driving mutations as well as clonal markers for the assessment of minimal residual disease are not always identifiable. Hence, there is a clinical need to further refine the genetic landscape of T-ALL including previously unrecognized fusion partners of commonly translocated genes in T-ALL of childhood. RESULTS: In this study, we screened n = 229 T-ALL cases by our targeted genomic capture high-throughput sequencing (gc-HTS) approach. In total, we identified n = 60 gene–gene fusions, present in n = 57 (25%) of the patients. Nine rare or even unrecognized translocations were identified and validated. Furthermore, owing to its interesting chromosomal structure, we studied the oncogenic potential of the complex rearrangement of chromosome 2 and 12, found in a near-early T-cell progenitor (ETP) ALL that leads to the fusion events PRKCE::ETV6 and ETV6::INO80D. Exogenous expression of PRKCE::ETV6 in Ba/F3 pro-B and D1 T-cells caused interleukin-independent proliferation and enhanced survival upon interleukin withdrawal, respectively. CONCLUSION: Our study underlines the heterogenous mutational landscape in T-ALL. The previously unrecognized PRKCE::ETV6 resulting from a complex rearrangement involving chromosome 2 and 12 demonstrated transforming potential in cytokine-dependent cellular models support the notion of a driver mutation in near ETP-ALL. Our data reconfirm the relevance of ETV6-fusion proteins in the pathogenesis of undifferentiated T-ALL. Importantly, genomic breakpoints at the ETV6 locus represent potentially robust MRD markers for (near) ETP-ALL that lack IG/TR rearrangements.

ETV6::INO80D↗

PubMed2027

Screening of Antiviral Agents Against CHIKV Using Reporter Virus.

Chikungunya virus (CHIKV) causes a disease characterized by chronic musculoskeletal inflammation for which specific antivirals are not yet available. Currently, a supportive therapy to alleviate fever and pain is used, but it does not limit viral replication or the persistence of chronic arthritis symptoms. Thus, the identification and development of new active molecules against CHIKV is urgently needed. Here, we present a cell-based methodology that enables the implementation of a rapid and cost-effective strategy for high- and medium-throughput screening (HTS) of compounds, including repurposed drugs or novel molecules. This methodology allows for the identification of novel antiviral hits with a good activity and selectivity profile against CHIKV.

Antiviral Agents↗

PubMed2026

Characterization of yam virus X isolates from Dioscorea trifida in Brazil.

OBJECTIVE: Yam virus X (YVX; Potexvirus ecsdioscoreae) is a positive-sense, flexuous RNA virus belonging to the family Alphaflexiviridae. It has been first reported from Guadeloupe, a French archipelago located in the Caribbean Sea. In this study, we investigated the virome in yam (Dioscorea spp.) plant material collected in the state of Bahia (Brazil) by high-throughput sequencing (HTS) on Illumina platform. The objective of the investigation was to explore the occurrence of YVX in yam from South America, and to study its genetic diversity compared to the only one YVX genome sequence available in the GenBank public database. RESULTS: An initial investigation by HTS of bulked RNA extracts (n=23, combined into 4 pools) revealed occurrence of YVX only in samples collected in the region of Valença. Subsequent screening by RT-PCR of the individual samples composing the pool uncovered infection with YVX only in Discorea trifida. Total RNA extracts from three infected plants were individually sequenced, resulting in the assembly of three complete genome sequences of YVX, showing ~84% nucleotide identity to the reference sequence from Guadeloupe. Our results contribute to expanding the pool of sequences available for YVX, supporting detection purposes and stimulating additional investigations for future studies on YVX diversity and evolution.

Brazil↗