scholarly journals In vivo recording of adult zebrafish electrocardiogram and assessment of drug-induced QT prolongation

2006 ◽  
Vol 291 (1) ◽  
pp. H269-H273 ◽  
Author(s):  
David J. Milan ◽  
Ian L. Jones ◽  
Patrick T. Ellinor ◽  
Calum A. MacRae
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Larval Fish ◽  
Low Noise ◽  
Qt Prolongation ◽  
Motion Artifacts ◽  
Adult Zebrafish ◽  
Perfusion System ◽  
Drug Induced

In the last decade the zebrafish has become a major model organism for the study of development and organogenesis. To maximize the experimental utility of this organism, it will be important to establish methods for adult phenotyping. We previously proposed that the embryonic zebrafish may be useful in high-throughput screening for drug-induced cardiotoxicity. We now describe a method for the reproducible recording of the adult zebrafish ECG and illustrate its application in the investigation of QT-prolonging drugs. Zebrafish ECGs were obtained by inserting two needle electrodes through the ventral epidermis. Fish were perfused orally, and motion artifacts were eliminated with a paralytic dose of μ-conotoxin GIIIB. Test compounds were delivered via the perfusion system. Without a means of hydration and oxygenation, the fish succumb rapidly. The use of a perfusion system allowed stable recording for >6 h. Baseline conduction intervals were as follows: PR, 66 ms (SD 14); QRS, 34 ms (SD 11); QT, 242 ms (SD 54); and R-R, 398 ms (SD 77). The known QT-prolonging agents astemizole, haloperidol, pimozide, and terfenadine caused corrected QT increases of 18% (SD 9), 16% (SD 11), 17% (SD 9), and 11% (SD 6), respectively. The control drugs clonidine, penicillin and propranolol did not prolong the corrected QT interval. In conclusion, perfusion and muscular paralysis allows stable, low-noise recording of zebrafish ECGs. Agents known to cause QT prolongation in humans caused QT prolongation in fish in each case. The development of rigorous tools for the phenotyping of adult zebrafish will complement the high-throughput assays currently under development for embryonic and larval fish.

scholarly journals A Multi-Omics Human Liver Organoid Screening Platform for DILI Risk Prediction

2021 ◽  
Author(s):  
Charles J. Zhang ◽  
Matthew J. O’Meara ◽  
Sophia R. Meyer ◽  
Sha Huang ◽  
Meghan M. Capeling ◽  
...  
Keyword(s):  
Drug Development ◽  
Risk Prediction ◽  
High Throughput ◽  
High Throughput Screening ◽  
Human Liver ◽  
Drug Induced ◽  
Predictive Capacity ◽  
On Chip

AbstractBackground and AimsDrug-induced liver injury (DILI) is a prominent failure mode in drug development resulting in clinical trial failures and post-approval withdrawal. Improved in vitro models for DILI risk prediction that can model diverse genetics are needed to improve safety and reduce high attrition rates in drug development. In this study, we evaluated the utility of human liver organoids (HLOs) for high-throughput DILI risk prediction and in an organ-on-chip system. The recent clinical failure of inarigivir soproxil due to DILI underscores the need for improved models.MethodsHLOs were adapted for high-throughput drug screening in dispersed-cell 384-well format and a collection of DILI-associated drugs were screened. HLOs were also adapted to a liver-chip system to investigate enhanced in vivo-like function. Both platforms were benchmarked for their ability to predict DILI using combined biochemical assays, microscopy-based morphological profiling, and transcriptomics.ResultsDispersed HLOs retained DILI predictive capacity of intact HLOs and are amenable to high-throughput screening allowing for measurable IC50 values for cytotoxicity. Distinct morphological differences were observed in cells treated with drugs exerting differing mechanisms of action. HLOs on chips were shown to increase albumin production, CYP450 expression and also release ALT/AST when treated with known DILI drugs. Importantly, HLO liver chips were able to predict hepatotoxicity of tenofovir-inarigivir and showed steatosis and mitochondrial perturbation via phenotypic and transcriptomic analysis.ConclusionsThe high throughput and liver-on-chip system exhibit enhanced in vivo-like function and demonstrate the utility of the platforms in early and late-stage drug development. Tenofovir-inarigivr associated hepatotoxicity was observed and highly correlates with the clinical manifestation of DILI.

scholarly journals High-throughput screening and rational design of biofunctionalized surfaces with optimized biocompatibility and antimicrobial activity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhou Fang ◽  
Junjian Chen ◽  
Ye Zhu ◽  
Guansong Hu ◽  
Haoqian Xin ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Rational Design ◽  
Click Reaction ◽  
Reaction Times ◽  
Great Promise ◽  
Biomaterial Surfaces

AbstractPeptides are widely used for surface modification to develop improved implants, such as cell adhesion RGD peptide and antimicrobial peptide (AMP). However, it is a daunting challenge to identify an optimized condition with the two peptides showing their intended activities and the parameters for reaching such a condition. Herein, we develop a high-throughput strategy, preparing titanium (Ti) surfaces with a gradient in peptide density by click reaction as a platform, to screen the positions with desired functions. Such positions are corresponding to optimized molecular parameters (peptide densities/ratios) and associated preparation parameters (reaction times/reactant concentrations). These parameters are then extracted to prepare nongradient mono- and dual-peptide functionalized Ti surfaces with desired biocompatibility or/and antimicrobial activity in vitro and in vivo. We also demonstrate this strategy could be extended to other materials. Here, we show that the high-throughput versatile strategy holds great promise for rational design and preparation of functional biomaterial surfaces.

scholarly journals A Microfluidic Spheroid Culture Device with a Concentration Gradient Generator for High-Throughput Screening of Drug Efficacy

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3355 ◽  
Author(s):  
Wanyoung Lim ◽  
Sungsu Park
Keyword(s):  
High Throughput ◽  
Concentration Gradient ◽  
High Throughput Screening ◽  
3D Cell Culture ◽  
Drug Efficacy ◽  
Cancer Drug ◽  
Spheroid Culture ◽  
Gradient Generator ◽  
Micro Channels

Three-dimensional (3D) cell culture is considered more clinically relevant in mimicking the structural and physiological conditions of tumors in vivo compared to two-dimensional cell cultures. In recent years, high-throughput screening (HTS) in 3D cell arrays has been extensively used for drug discovery because of its usability and applicability. Herein, we developed a microfluidic spheroid culture device (μFSCD) with a concentration gradient generator (CGG) that enabled cells to form spheroids and grow in the presence of cancer drug gradients. The device is composed of concave microwells with several serpentine micro-channels which generate a concentration gradient. Once the colon cancer cells (HCT116) formed a single spheroid (approximately 120 μm in diameter) in each microwell, spheroids were perfused in the presence of the cancer drug gradient irinotecan for three days. The number of spheroids, roundness, and cell viability, were inversely proportional to the drug concentration. These results suggest that the μFSCD with a CGG has the potential to become an HTS platform for screening the efficacy of cancer drugs.

scholarly journals A High-Throughput Cellular Screening Assay for Small-Molecule Inhibitors and Activators of Cytoplasmic Dynein-1-Based Cargo Transport

2020 ◽  
Vol 25 (9) ◽  
pp. 985-999
Author(s):  
John Vincent ◽  
Marian Preston ◽  
Elizabeth Mouchet ◽  
Nicolas Laugier ◽  
Adam Corrigan ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Cytoplasmic Dynein ◽  
Lead Discovery ◽  
Screening Assay ◽  
Age Related ◽  
The Uk

Cytoplasmic dynein-1 (hereafter dynein) is a six-subunit motor complex that transports a variety of cellular components and pathogens along microtubules. Dynein’s cellular functions are only partially understood, and potent and specific small-molecule inhibitors and activators of this motor would be valuable for addressing this issue. It has also been hypothesized that an inhibitor of dynein-based transport could be used in antiviral or antimitotic therapy, whereas an activator could alleviate age-related neurodegenerative diseases by enhancing microtubule-based transport in axons. Here, we present the first high-throughput screening (HTS) assay capable of identifying both activators and inhibitors of dynein-based transport. This project is also the first collaborative screening report from the Medical Research Council and AstraZeneca agreement to form the UK Centre for Lead Discovery. A cellular imaging assay was used, involving chemically controlled recruitment of activated dynein complexes to peroxisomes. Such a system has the potential to identify molecules that affect multiple aspects of dynein biology in vivo. Following optimization of key parameters, the assay was developed in a 384-well format with semiautomated liquid handling and image acquisition. Testing of more than 500,000 compounds identified both inhibitors and activators of dynein-based transport in multiple chemical series. Additional analysis indicated that many of the identified compounds do not affect the integrity of the microtubule cytoskeleton and are therefore candidates to directly target the transport machinery.

scholarly journals A cell-based high-throughput screen identifies drugs that cause bleeding disorders by off-targeting the vitamin K cycle

Blood ◽  
2020 ◽  
Vol 136 (7) ◽  
pp. 898-908
Author(s):  
Xuejie Chen ◽  
Caihong Li ◽  
Da-Yun Jin ◽  
Brian Ingram ◽  
Zhenyu Hao ◽  
...  
Keyword(s):  
Vitamin K ◽  
High Throughput ◽  
High Throughput Screening ◽  
Molecular Mechanisms ◽  
Coagulation Factors ◽  
Bleeding Complications ◽  
Bleeding Disorders ◽  
Drug Induced ◽  
Vitamin K Cycle ◽  
Established Cell

Abstract Drug-induced bleeding disorders contribute to substantial morbidity and mortality. Antithrombotic agents that cause unintended bleeding of obvious cause are relatively easy to control. However, the mechanisms of most drug-induced bleeding disorders are poorly understood, which makes intervention more difficult. As most bleeding disorders are associated with the dysfunction of coagulation factors, we adapted our recently established cell-based assay to identify drugs that affect the biosynthesis of active vitamin K–dependent (VKD) coagulation factors with possible adverse off-target results. The National Institutes of Health (NIH) Clinical Collection (NCC) library containing 727 drugs was screened, and 9 drugs were identified, including the most commonly prescribed anticoagulant warfarin. Bleeding complications associated with most of these drugs have been clinically reported, but the pathogenic mechanisms remain unclear. Further characterization of the 9 top-hit drugs on the inhibition of VKD carboxylation suggests that warfarin, lansoprazole, and nitazoxanide mainly target vitamin K epoxide reductase (VKOR), whereas idebenone, clofazimine, and AM404 mainly target vitamin K reductase (VKR) in vitamin K redox cycling. The other 3 drugs mainly affect vitamin K availability within the cells. The molecular mechanisms underlying the inactivation of VKOR and VKR by these drugs are clarified. Results from both cell-based and animal model studies suggest that the anticoagulation effect of drugs that target VKOR, but not VKR, can be rescued by the administration of vitamin K. These findings provide insights into the prevention and management of drug-induced bleeding disorders. The established cell-based, high-throughput screening approach provides a powerful tool for identifying new vitamin K antagonists that function as anticoagulants.

scholarly journals In Vivo, High-Throughput Selection of Thermostable Cyclohexanone Monooxygenase (CHMO)

Catalysts ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 935
Author(s):  
Sarah Maxel ◽  
Linyue Zhang ◽  
Edward King ◽  
Ana Paula Acosta ◽  
Ray Luo ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Rational Design ◽  
Random Mutagenesis ◽  
Residual Activity ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Coli Strain ◽  
Screening Tools ◽  
Cyclohexanone Monooxygenase

Cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871 is characterized as having wide substrate versatility for the biooxidation of (cyclic) ketones into esters and lactones with high stereospecificity. Despite industrial potential, CHMO usage is restricted by poor thermostability. Limited high-throughput screening tools and challenges in rationally engineering thermostability have impeded CHMO engineering efforts. We demonstrate the application of an aerobic, high-throughput growth selection platform in Escherichia coli (strain MX203) for the discovery of thermostability enhancing mutations for CHMO. The selection employs growth for the easy readout of CHMO activity in vivo, by requiring nicotinamide adenine dinucleotide phosphate (NADPH)-consuming enzymes to restore cellular redox balance. In the presence of the native substrate cyclohexanone, variant CHMO GV (A245G-A288V) was discovered from a random mutagenesis library screened at 42 °C. This variant retained native activity, exhibited ~4.4-fold improvement in residual activity after 30 °C incubation, and demonstrated ~5-fold higher cyclohexanone conversion at 37 °C compared to the wild type. Molecular modeling indicates that CHMO GV experiences more favorable residue packing and supports additional backbone hydrogen bonding. Further rational design resulted in CHMO A245G-A288V-T415C with improved thermostability at 45 °C. Our platform for oxygenase evolution enabled the rapid engineering of protein stability critical for industrial scalability.

scholarly journals Meeting Report: High-Throughput Technologies for In Vivo Imaging Agents

2005 ◽  
Vol 4 (2) ◽  
pp. 153535002005051 ◽  
Author(s):  
Robert J. Gillies ◽  
John M. Hoffman ◽  
Kit S. Lam ◽  
Anne E. Menkens ◽  
David R. Piwnica-Worms ◽  
...  
Keyword(s):  
High Throughput ◽  
In Vivo Imaging ◽  
High Throughput Screening ◽  
Imaging Agents ◽  
Meeting Report ◽  
Screening Methods ◽  
Pharmacological Properties ◽  
Drug Candidates ◽  
Good Imaging

Combinatorial chemistry and high-throughput screening have become standard tools for discovering new drug candidates with suitable pharmacological properties. Now, those same technologies are starting to be applied to the problem of discovering novel in vivo imaging agents. Important differences in the biological and pharmacological properties needed for imaging agents, compared to those for a therapeutic agent, require new screening methods that emphasize those characteristics, such as optimized residence time and tissue specificity, that make for a good imaging agent candidate.

scholarly journals Phenotypic assays in yeast and zebrafish reveal drugs that rescue ATP13A2 deficiency

2019 ◽  
Vol 1 (1) ◽  
Author(s):  
Ursula Heins-Marroquin ◽  
Paul P Jung ◽  
Maria Lorena Cordero-Maldonado ◽  
Alexander D Crawford ◽  
Carole L Linster
Keyword(s):  
Heavy Metal ◽  
High Throughput ◽  
High Throughput Screening ◽  
Drug Testing ◽  
Neuronal Ceroid Lipofuscinosis ◽  
Drug Repurposing ◽  
In Vivo Model ◽  
Inherited Disorders ◽  
Zebrafish Model

Abstract Mutations in ATP13A2 (PARK9) are causally linked to the rare neurodegenerative disorders Kufor-Rakeb syndrome, hereditary spastic paraplegia and neuronal ceroid lipofuscinosis. This suggests that ATP13A2, a lysosomal cation-transporting ATPase, plays a crucial role in neuronal cells. The heterogeneity of the clinical spectrum of ATP13A2-associated disorders is not yet well understood and currently, these diseases remain without effective treatment. Interestingly, ATP13A2 is widely conserved among eukaryotes, and the yeast model for ATP13A2 deficiency was the first to indicate a role in heavy metal homeostasis, which was later confirmed in human cells. In this study, we show that the deletion of YPK9 (the yeast orthologue of ATP13A2) in Saccharomyces cerevisiae leads to growth impairment in the presence of Zn2+, Mn2+, Co2+ and Ni2+, with the strongest phenotype being observed in the presence of zinc. Using the ypk9Δ mutant, we developed a high-throughput growth rescue screen based on the Zn2+ sensitivity phenotype. Screening of two libraries of Food and Drug Administration-approved drugs identified 11 compounds that rescued growth. Subsequently, we generated a zebrafish model for ATP13A2 deficiency and found that both partial and complete loss of atp13a2 function led to increased sensitivity to Mn2+. Based on this phenotype, we confirmed two of the drugs found in the yeast screen to also exert a rescue effect in zebrafish—N-acetylcysteine, a potent antioxidant, and furaltadone, a nitrofuran antibiotic. This study further supports that combining the high-throughput screening capacity of yeast with rapid in vivo drug testing in zebrafish can represent an efficient drug repurposing strategy in the context of rare inherited disorders involving conserved genes. This work also deepens the understanding of the role of ATP13A2 in heavy metal detoxification and provides a new in vivo model for investigating ATP13A2 deficiency.

Sign in / Sign up

Export Citation Format

Share Document