Search Results (201)

Optical density (OD)-based cell growth measurement is commonly used in high-throughput screening (HTS) during drug discovery or when deciphering the pharmaceutical mechanism of action. While resuspending the cells via a mixing step is often assumed to be necessary prior to OD measurement, its essentiality in HTS workflows has not been systematically verified. Here, through the measurement of the growth of several strains of the microbial yeast Schizosaccharomyces pombe cells, we compared the overall growth dynamics between samples that have been mixed and not mixed. Using statistical quantification by a two-tailed paired t-test followed by multiple comparison corrections, we concluded from the comparison of the doubling time of cells growing in the exponential phase that mixing did not significantly affect the biological interpretation compared to unmixed samples. Doubling time quantification between mixed and unmixed samples showed a difference of approximately 10% on average based on the assessment of the growth of eight strains. As such, if the experimental outcome can accommodate this level of variability, incorporating a mixing step before OD determination would not be necessary. These observations support the simplification of HTS processes, improving the cost efficacy and process efficiency of readouts, yet maintaining the accuracy of data acquisition. Full article

The optimization of membrane permeability is a pivotal approach for mitigating late-stage failures in peptide drug development. By leveraging linker chemical diversity, stapled peptides utilize linker engineering to precisely modulate key physicochemical parameters—such as lipophilicity and conformational constraints—to overcome the desolvation energy penalty. This review systematically evaluates linker-based strategies for enhancing the permeability of stapled peptides, categorized into two primary dimensions: (1) high-throughput screening (HTS) compatibility, focusing on the integration of functionalized linkers into mRNA display, phage display, and DNA-encoded libraries (DELs) to identify lead scaffolds with inherent permeability potential during early discovery; and (2) post-screening structural refinement, covering rational design strategies including intramolecular hydrogen-bond (IMHB) shielding, “chameleonic” adaptations, and stimuli-responsive reversible stapling. Furthermore, we analyze the paradigm shift in assessment methodologies from qualitative imaging to quantitative cytosolic delivery assays, which have deepened our understanding of mechanisms such as the charge/lipophilicity threshold balance and metabolism-driven trapping. Overall, linker engineering provides a robust technical roadmap for developing the next generation of cell-permeable stapled peptide therapeutics. Full article

Background: Three-dimensional (3D) cancer models are currently essential tools in high-throughput screening (HTS), serving as a bridge between in vitro and in vivo approaches during drug development. Even though spheroids offer many advantages over 2D cultures, analyzing 3D cultures with heterogeneous morphology remains challenging due to the lack of standardized visualization techniques and multiparameter analysis. Methods: In this work, an optimized CellProfiler pipeline and a Python algorithm for weighting morphological features are used to visualize, extract, and analyze morphological data from spheroids derived from colorectal and pancreatic cancer cell lines with diverse morphologies (HCT116, LoVo, PANC-1, and CFPAC-1). Results: We developed a feature weighting process that combines multiple morphological parameters into a single metric using principal component analysis (PCA). There is a strong correlation between this process and a standard Alamar Blue proliferation assay (r = 0.89, ρ = 0.91, p < 0.001). Using this method, we were able to ascertain the IC₅₀ values of substances that did not produce results in cell lines with heterogeneous morphology (LoVo and CFPAC-1) using a standard proliferation assay. Conclusions: By removing the need for tracer dyes, the resulting methodology may lower costs while accelerating preclinical drug development through informative multiparameter analysis of compound efficacy. Full article

Background: Bicuspid aortic valve (BAV) is the most common congenital heart defect, and its complications (namely, dilatation of the thoracic ascending aorta) raise concerns regarding the proper timing of aortic surgery. The study aim is to unravel the genetic basis of BAV and its complications through a high-throughput sequencing (HTS) approach and segregation analysis if family members were available. Methods: Fifty-two Italian BAV patients were analyzed by HTS using the Illumina MiSeq platform. Targeted sequencing of 97 genes known to be or plausibly associated with connective tissue disorders or aorthopathy was performed. Thirty-five first-degree relatives of N = 10 probands underwent mutational screening for variants identified in the index cases. Results: HTS identified 194 rare (MAF < 0.01) variants in 63 genes. Regarding previously reported genes, five NOTCH1 variants in four BAV patients, four FBN1 variants in two patients and one GATA5 variant in one patient were identified. Interestingly, among further loci, the possible contribution of PDIA2, LRP1 and CAPN2 was suggested by (a) the increased prevalence of rare genetic variants, independently from their ACMG classification in the whole BAV cohort, and (b) segregation analyses of variants identified in family members. Moreover, the present data also suggest the possible contribution of rare variants to BAV complications, specifically MYLK in aortic dilatation, CAPN2 in BAV calcification and VHL and AGGF1 in valve stenosis. Conclusions: Our results underline clinical and genetic diagnosis complexity in traits considered monogenic, such as BAV, but characterized by variability in disease phenotypic expression (incomplete penetrance), as well as the contribution of different major and modifier genes to the development of complications. Full article

The development of new alternative models is essential to overcome the limitations of traditional two-dimensional (2D) cell cultures and animal models. Three-dimensional (3D) models, such as organoids, better mimic the structural and functional complexity of mammalian organs, thereby reducing the ethical and economic issues related to animal experimentation. These systems provide more physiologically relevant environments, improving the accuracy of disease modeling and drug response prediction. In this context, we have developed mouse hepatic organoids from livers of adult BALB/c mice and characterized them by microscopy and transcriptional analysis. This model was applied to a robust and reproducible high-throughput screening (HTS) platform for testing cytotoxicity at the preclinical stage of drug discovery. In addition, mouse hepatic organoids were co-cultured with amastigotes of Leishmania donovani parasites to establish a model of host–parasite interaction, which was characterized by RNA-seq linked to differential expression analysis and cytokine production by the hepatic organoids. The findings provided in this work establish mouse hepatic organoids as an alternative model for drug discovery and pathogenesis studies. Full article

The widespread application of nanoparticles (NPs) in fields ranging from consumer products to industrial processes has led to increased concerns about their potential toxic effects on human health and the environment. While traditional toxicological studies often evaluate the effects of individual NPs, real-world exposure scenarios typically involve mixtures of NPs, where interactions between particles can significantly alter their toxicological profiles. This study provides an overview of overcoming this gap by possible utilization of high-throughput screening (HTS) for evaluation of the combined effects of NP mixtures under various exposure conditions. This review discusses HTS of metal oxide NPs, which have cytotoxic, genotoxic, and oxidative stress-inducing effects. Using HTS, this review describes multiple studies with multiple mixture ratios and exposure durations using human lung epithelial cells and zebrafish embryo systems. The review also describes a range of interactions, from synergistic effects, where the combined toxicity might be the sum of individual toxicities. Oxidative stress and metal ion release were key drivers of toxicity, particularly in metal oxide-dominant NP mixtures. This theoretical study highlights the importance of integrating HTS into nanotoxicology research to provide a more comprehensive understanding of the toxic behavior of NPs. Full article

Over the past two decades, three-dimensional cell culture (3DCC) and bioprinting (3DBP) technology have been at the forefront of developing engineered human cardiac tissue. Compared to 2D culture systems, 3DCC-based models more accurately replicate key characteristics of living tissues such as diffusion dynamics, interactions between cells and the extracellular matrix, as well as the presence of supporting stromal components. The rise of new 3DBP techniques serves to facilitate the robust and rapid generation of 3D tissue alongside real-time assessment of their characteristics. In order to capitalize on their translational potential, high-throughput screening (HTS) is required as research draws nearer to replicating clinical trials with cardiovascular-bioprinted tissues, and testing for the safety and efficacy of novel therapeutics. In this review, we summarize trending cardiac tissue models, as well as the state of their related or integrated HTS process and development. With an emphasis on the need for scale-up, compatibility, and standardization in HTS, the coalescence of 3DBP models and screening can provide improved disease modeling, drug efficacy, and toxicity testing. Full article

High-throughput screening (HTS) is an essential process in drug discovery, requiring platforms that ensure reagent economy, high efficiency, and resistance to cross-contamination. Click chemistry is well suited for HTS because of its biocompatibility, high selectivity, and quantitative fluorescent readout. We focus on droplet-array sandwiching technology (DAST), in which two droplet microarrays (DMAs) are vertically opposed to achieve solute transport and reagent mixing by controlled contact and separation. Herein, we integrate click chemistry with DAST and evaluate its feasibility as a HTS platform. In DAST, DMAs are formed on wettability-patterned (WP; hydrophilic/hydrophobic) substrates, preserving resistance to cross-contamination. First, we immobilized dibenzocyclooctyne (DBCO) on a WP substrate and verified the occurrence of DBCO–azide reaction using an azide-functional fluorescent dye. The fluorescence intensity increased with concentration and reached a plateau at higher concentrations, indicating saturation behavior in the DBCO–azide click reaction. Second, acoustic mixing with repeated droplet contact–separation was applied to generate concentration gradients on a single substrate while maintaining droplet independence. Third, we qualitatively reproduced the expected concentration dependence of manual handling by combining DAST-based gradient formation with click reaction fluorescence readout. These results reveal that DAST enables a reagent-efficient, cross-contamination-resistant, and low-instrument-dependent HTS foundation for click-chemistry-based assays. Full article

Viral diseases pose a significant threat to lily (Lilium spp.) cultivation; however, large-scale assessments of virus prevalence and diversity in South Korea are limited. This study combined RT-PCR surveys, high-throughput sequencing (HTS), and analyses of 48 lily hybrid transcriptomes to characterize the lily virome. RT-PCR screening of 100 samples from 13 regions showed that 87% were infected, primarily with lily mottle virus (LMoV, 65%), Plantago asiatica mosaic virus (PlAMV, 34%), cucumber mosaic virus (CMV, 34%), and lily symptomless virus (LSV, 25%). Mixed infections were approximately twice as frequent as single infections and were associated with greater symptom severity, particularly in triple-virus combinations. High-throughput sequencing expanded detection to six viruses, including milk vetch dwarf virus (MDV) and lily virus B (LVB), the latter confirmed as a variant of strawberry latent ringspot virus (SLRSV). Near-complete genomes of several viruses were assembled and validated through RT-PCR. Transcriptome mining identified eight virus species across 26 cultivars; PlAMV was the most common, and viral loads varied significantly among hybrids. Phylogenetic analyses revealed close relationships between Korean and Chinese isolates and host-related clustering in PlAMV. These findings highlight the complexity of lily viromes in South Korea and provide essential resources for diagnostics, disease management, and biosecurity. Full article

Background: Prostate cancer (PCa) is the primary contributor to male cancer-related mortality and currently lacks effective treatment options. The Modified Guizhi Fuling Decoction (MGFD) is used in clinical practice to treat multiple tumors. This research focused on the mechanisms of action (MOA) in MGFD that inhibit PCa. Methods: The impact of MGFD on PCa cells (PC3 and DU145) was examined via Cell Counting Kit-8, wound healing assays, and transwell assays. To determine the MOA, high-throughput sequencing based high-throughput screening (HTS²) was utilized along with network pharmacology. Results: The findings indicated that MGFD suppressed the proliferation, migration, and invasion of PCa cells. We then utilized the HTS² assay to generate 270 gene expression profiles from PCa cells perturbed by MGFD. Large-scale transcriptional analysis highlighted three pathways closely associated with PCa: the TNF signaling pathway, cellular senescence, and FoxO signaling pathway. Through the combination of network pharmacology and bioinformatics, we discovered four primary targets through which MGFD acts on PCa: AKT serine/threonine kinase 1 (AKT1), Caspase-8 (CASP8), Cyclin-Dependent Kinase 1 (CDK1), and Cyclin D1 (CCND1). Finally, molecular docking demonstrated that the potential bioactive compounds baicalein, quercetin, and 5-[[5-(4-methoxyphenyl)-2-furyl] methylene] barbituric acid strongly bind to CDK1, AKT1, and CASP8, respectively. Conclusions: This research shows that MGFD displays encouraging anticancer effects via various mechanisms. Its multi-target activity profile underscores its promise as a potential therapeutic option for PCa treatment and encourages additional in vivo validation studies. Full article

Integrase is a key protein during HIV-1 replication as it catalyzes the integration of viral DNA into the host DNA. After several decades of research, highly potent and selective active site inhibitors have emerged. The new challenge is now to develop molecules with an original mode of action, targeting integrase out of its catalytic site. During a previous study, we developed an in vitro assay to monitor the interaction between HIV-1 integrase and one of its cellular partners, GCN2. This AlphaLISA-based assay was validated as a platform for chemical modulator screening. In the present study, we used a library of natural products from the Developmental Therapeutics Program (NIH) to identify novel chemical leads. The best modulators were characterized and a structure–activity relationship study was initiated with a limited number of derivatives. We found that most inhibitors were tricylic or tetraclyclic molecules, with the most potent belonging to the anthracyclines/anthraquinones. Of note, several molecules exhibited interesting cellular activities and may be suitable for further optimization. Full article

The development of molecules that interact with G-quadruplex (G4) sequences requires effective evaluation methods. Several techniques are currently available, including nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and mass spectrometry (MS), fluorescence using FRET-melting, G4-fluorescent intercalator displacement assay (G4-FID) and affinity chromatography. Among these, CD spectroscopy is gaining prominence due to its lower material requirements, faster experimentation and quicker data processing. However, conventional CD methods have limitations, such as higher sample volume required and the inability to handle high-throughput analysis efficiently. The use of synchrotron radiation in high-throughput analysis methods (HT-SRCD) has further advanced the investigation of small-molecule interactions with DNA G4 structures in the presence of various monovalent cations. HT-SRCD offers the capability to analyze multiple samples simultaneously, overcoming the limitations of conventional CD methods. To validate this approach, three biologically relevant G4 sequences—HTelo1, G3T3 and T95-2T—were investigated. Their interactions with a library of small tetrazole-based molecules, synthesized via a four-component Ugi reaction, and with a peptide sequence deriving from RHAU helicases (Rhau25), were evaluated. The results demonstrate that this method not only effectively discriminates between different ligands but also provides valuable insights into the selectivity and the modes of interaction of these ligands with the G4 sequences. Full article

Neurodegenerative diseases (NDDs) that are characterized by the accumulation of alpha-synuclein (α-syn) aggregates in both neurons and the non-neuronal cells of the brain are called synucleinopathies. The most common synucleinopathies includes Parkinson’s disease (PD), Parkinson’s disease dementia (PDD), multiple system atrophy (MSA), and dementia with Lewy bodies (DLB). Significant progress has been made in the development of positron emission tomography (PET) radiotracers for synucleinopathies, yielding several α-syn tracers that have entered clinical studies. However, selective α-syn imaging still faces inherent challenges. This review provides a comprehensive overview of the progress in α-syn PET radiotracers from three angles: Alzheimer’s disease (AD)-derived scaffolds, representative compound scaffolds and analogs, and the identification of α-syn tracers through high-throughput screening (HTS). We discuss the characteristics, advantages, and limitations of the tracers for preclinical and clinical application. Finally, future directions in the development of radioligands for proteinopathies are discussed. There is no clinical available PET radiotracer for imaging α-syn aggregates, but these advances have laid a key foundation for non-invasive α-syn imaging and early diagnosis of synucleinopathies. Full article

Brain organoids are self-organized, three-dimensional (3D) aggregates derived from human embryonic stem cells, induced pluripotent stem cells, or primary organs with cell types and cellular architectures resembling those of the developing human brain. Recent studies have shown the use of region-specific brain organoids for modeling various diseases ranging from neurodevelopmental and neurodegenerative diseases to different brain cancers, which have numerous applications in fundamental research and the development of new drugs, personalized treatment, and regenerative medicine. Consequently, the use of brain organoids in drug discovery is complex and challenging and still an emerging area in this field. This review article summarizes the primary stem cells used in brain organoid generation, region-specific brain organoids, and the functional assays used in their characterization. In addition, we discuss the use of brain organoids in modeling neurodevelopmental and neurodegenerative diseases and pediatric brain cancers, as well as the application of organoids, assembloids, and tumoroids in cancer neuroscience. We further explore the recent advances in using brain organoids in high-throughput screening to improve their use for drug discovery. Full article

Background: The 2C protein of foot-and-mouth disease virus (FMDV), a member of helicase superfamily 3 (SF3), drives viral genome replication and serves as a critical target for antiviral drug development. Methods: A fluorescence resonance energy transfer (FRET)-based high-throughput screening (HTS) platform was developed to identify 2C helicase inhibitors. Primary screening evaluated 4424 compounds for helicase inhibition. Molecular docking analyzed inhibitor interactions with the N207 residue within the catalytic core and helicase inhibition assays classified the inhibitor type (mixed, competitive, noncompetitive). Differential scanning fluorimetry (nanoDSF) quantified 2C thermal destabilization. Antiviral activity was assessed via indirect immunofluorescence, RT-qPCR, and plaque reduction assays. Results: Six compounds inhibited 2C helicase activity at >620 μM. Molecular docking revealed hydrogen bonding, hydrophobic interactions, and π-cation stabilization at the catalytic core. 2-MPO and MPPI were classified as mixed-type inhibitors, 5-TzS⁻ and 2-PyOH as competitive, and DCMQ/Spiro-BD-CHD-dione as noncompetitive. NanoDSF showed a ΔT_m ≥ 1.5 °C (2.5 mM compounds), with reduced destabilization in N207A mutants. Antiviral assays identified 2-MPO and MPPI as optimal inhibitors. MPPI achieved effective FMDV suppression at 160 μM, exhibiting two orders of magnitude higher potency than 2-MPO (400 μM). Conclusions: The established FRET-based HTS platform targeting 2C helicase facilitates anti-FMDV lead discovery, while 2C inhibitors may serve as an effective therapeutic strategy against other picornaviruses. Full article