scholarly journals A Novel Multiparametric Drug-Scoring Method for High-Throughput Screening of 3D Multicellular Tumor Spheroids Using the Celigo Image Cytometer

2017 ◽  
Vol 22 (5) ◽  
pp. 547-557 ◽  
Author(s):  
Scott Cribbes ◽  
Sarah Kessel ◽  
Scott McMenemy ◽  
Jean Qiu ◽  
Leo Li-Ying Chan
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method ◽  
Three Dimensional ◽  
Cancer Drug ◽  
Multicellular Tumor Spheroids ◽  
Tumor Spheroids ◽  
Scoring Method ◽  
Drug Candidates ◽  
Drug Compounds

Three-dimensional (3D) tumor models have been increasingly used to investigate and characterize cancer drug compounds. The ability to perform high-throughput screening of 3D multicellular tumor spheroids (MCTS) can highly improve the efficiency and cost-effectiveness of discovering potential cancer drug candidates. Previously, the Celigo Image Cytometer has demonstrated a novel method for high-throughput screening of 3D multicellular tumor spheroids. In this work, we employed the Celigo Image Cytometer to examine the effects of 14 cancer drug compounds on 3D MCTS of the glioblastoma cell line U87MG in 384-well plates. Using parameters such as MCTS diameter and invasion area, growth and invasion were monitored for 9 and 3 d, respectively. Furthermore, fluorescent staining with calcein AM, propidium iodide, Hoechst 33342, and caspase 3/7 was performed at day 9 posttreatment to measure viability and apoptosis. Using the kinetic and endpoint data generated, we created a novel multiparametric drug-scoring system for 3D MCTS that can be used to identify and classify potential drug candidates earlier in the drug discovery process. Furthermore, the combination of quantitative and qualitative image data can be used to delineate differences between drugs that induce cytotoxic and cytostatic effects. The 3D MCTS-based multiparametric scoring method described here can provide an alternative screening method to better qualify tested drug compounds.

scholarly journals Real-Time Apoptosis and Viability High-Throughput Screening of 3D Multicellular Tumor Spheroids Using the Celigo Image Cytometer

2017 ◽  
Vol 23 (2) ◽  
pp. 202-210 ◽  
Author(s):  
Sarah Kessel ◽  
Scott Cribbes ◽  
Surekha Bonasu ◽  
Jean Qiu ◽  
Leo Li-Ying Chan
Keyword(s):  
Real Time ◽  
High Throughput ◽  
High Throughput Screening ◽  
Cancer Drug ◽  
Tumor Spheroid ◽  
Potential Drug ◽  
Multicellular Tumor Spheroid ◽  
Drug Candidates ◽  
Secondary Screen ◽  
Drug Compounds

Three-dimensional tumor spheroid models have been increasingly used to investigate and characterize cancer drug compounds. Previously, the Celigo image cytometer has demonstrated its utility in a high-throughput screening manner for evaluating potential drug candidates in a 3D multicellular tumor spheroid (MCTS) primary screen. In addition, we have developed real-time kinetic caspase 3/7 apoptosis and propidium iodide viability 3D MCTS assays, both of which can be used in a secondary screen to better characterize the hit compounds. In this work, we monitored the kinetic apoptotic and cytotoxic effects of 14 compounds in 3D MCTS produced from the glioblastoma cell line U87MG in 384-well plates for 9 days. The kinetic results allowed the categorization of the effects from 14 drug compounds into early and late cytotoxic, apoptotic, cytostatic, and no effects. The real-time apoptosis and viability screening method can serve as an improved secondary screen to better understand the mechanism of action of these potential drug candidates identified from the primary screen, allowing one to identify a more qualified drug candidate and streamline the drug discovery process of research and development.

scholarly journals High-Throughput 3D Tumor Spheroid Screening Method for Cancer Drug Discovery Using Celigo Image Cytometry

2016 ◽  
Vol 22 (4) ◽  
pp. 454-465 ◽  
Author(s):  
Sarah Kessel ◽  
Scott Cribbes ◽  
Olivier Déry ◽  
Dmitry Kuksin ◽  
Eric Sincoff ◽  
...  
Keyword(s):  
High Throughput ◽  
Dose Response ◽  
High Throughput Screening ◽  
Screening Method ◽  
Cancer Drug ◽  
Seeding Density ◽  
Tumor Spheroid ◽  
In Vivo Models ◽  
Drug Candidates

Oncologists have investigated the effect of protein or chemical-based compounds on cancer cells to identify potential drug candidates. Traditionally, the growth inhibitory and cytotoxic effects of the drugs are first measured in 2D in vitro models, and then further tested in 3D xenograft in vivo models. Although the drug candidates can demonstrate promising inhibitory or cytotoxicity results in a 2D environment, similar effects may not be observed under a 3D environment. In this work, we developed an image-based high-throughput screening method for 3D tumor spheroids using the Celigo image cytometer. First, optimal seeding density for tumor spheroid formation was determined by investigating the cell seeding density of U87MG, a human glioblastoma cell line. Next, the dose–response effects of 17-AAG with respect to spheroid size and viability were measured to determine the IC50 value. Finally, the developed high-throughput method was used to measure the dose response of four drugs (17-AAG, paclitaxel, TMZ, and doxorubicin) with respect to the spheroid size and viability. Each experiment was performed simultaneously in the 2D model for comparison. This detection method allowed for a more efficient process to identify highly qualified drug candidates, which may reduce the overall time required to bring a drug to clinical trial.

scholarly journals Supercomputer-aided Drug Repositioning at Scale: Virtual Screening for SARS-CoV-2 Protease Inhibitor

2020 ◽  
Author(s):  
Sangjae Seo ◽  
Jung Woo Park ◽  
Dosik An ◽  
Junwon Yoon ◽  
Hyojung Paik ◽  
...  
Keyword(s):  
Virtual Screening ◽  
Protease Inhibitor ◽  
Binding Affinity ◽  
High Throughput ◽  
Active Site ◽  
High Throughput Screening ◽  
Drug Repositioning ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Drug Candidates

Coronavirus diseases (COVID-19) outbreak has been labelled a pandemic. For the prioritization of treatments to cope with COVID-19, it is important to conduct rapid high-throughput screening of chemical compounds to repurposing the approved drugs, such as repositioning of chloroquine (Malaria drug) for COVID-19. In this study, exploiting supercomputer resource, we conducted high-throughput virtual screening for potential repositioning candidates of the protease inhibitor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Using the three dimensional structure of main protease (Mpro) of SARS-CoV-2, we evaluated binding affinity between Mpro and drug candidates listed in SWEETLEAD library and ChEMBL database. Docking scores of 19,168 drug molecules at the active site of Mpro were calculated using Autodock Vina package. Among the calculated result, we selected 43 drug candidates and ran molecular dynamics (MD) simulation to further investigate protein-drug interaction. Among compounds that bind to the active site of SARS-CoV-2, we finally selected the 8 drugs showing the highest binding affinity; asunaprevir, atazanavir, dasabuvir, doravirine, fosamprenavir, ritonavir, voxilaprevir and amprenavir, which are the antiviral drugs of hepatitis C virus or human immunodeficiency virus. We expect that the present study provides comprehensive insights into the development of antiviral medication, especially for the treatment of COVID-19.<div><br></div><div>* Attached excel file contains a full list of results of docking calculations</div>

scholarly journals Supercomputer-aided Drug Repositioning at Scale: Virtual Screening for SARS-CoV-2 Protease Inhibitor

2020 ◽  
Author(s):  
Sangjae Seo ◽  
Jung Woo Park ◽  
Dosik An ◽  
Junwon Yoon ◽  
Hyojung Paik ◽  
...  
Keyword(s):  
Virtual Screening ◽  
Protease Inhibitor ◽  
Binding Affinity ◽  
High Throughput ◽  
Active Site ◽  
High Throughput Screening ◽  
Drug Repositioning ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Drug Candidates

Coronavirus diseases (COVID-19) outbreak has been labelled a pandemic. For the prioritization of treatments to cope with COVID-19, it is important to conduct rapid high-throughput screening of chemical compounds to repurposing the approved drugs, such as repositioning of chloroquine (Malaria drug) for COVID-19. In this study, exploiting supercomputer resource, we conducted high-throughput virtual screening for potential repositioning candidates of the protease inhibitor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Using the three dimensional structure of main protease (Mpro) of SARS-CoV-2, we evaluated binding affinity between Mpro and drug candidates listed in SWEETLEAD library and ChEMBL database. Docking scores of 19,168 drug molecules at the active site of Mpro were calculated using Autodock Vina package. Among the calculated result, we selected 43 drug candidates and ran molecular dynamics (MD) simulation to further investigate protein-drug interaction. Among compounds that bind to the active site of SARS-CoV-2, we finally selected the 8 drugs showing the highest binding affinity; asunaprevir, atazanavir, dasabuvir, doravirine, fosamprenavir, ritonavir, voxilaprevir and amprenavir, which are the antiviral drugs of hepatitis C virus or human immunodeficiency virus. We expect that the present study provides comprehensive insights into the development of antiviral medication, especially for the treatment of COVID-19.<div><br></div><div>* Attached excel file contains a full list of results of docking calculations</div>

Directed Evolution of P450 Fatty Acid Decarboxylases via High-Throughput Screening Towards Improved Catalytic Activity

2019 ◽  
Author(s):  
Huifang Xu ◽  
Weinan Liang ◽  
Linlin Ning ◽  
Yuanyuan Jiang ◽  
Wenxia Yang ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Fatty Acid ◽  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
Colorimetric Detection ◽  
Screening Method ◽  
Random Mutagenesis ◽  
Direct Production ◽  
Consumption Activity

P450 fatty acid decarboxylases (FADCs) have recently been attracting considerable attention owing to their one-step direct production of industrially important 1-alkenes from biologically abundant feedstock free fatty acids under mild conditions. However, attempts to improve the catalytic activity of FADCs have met with little success. Protein engineering has been limited to selected residues and small mutant libraries due to lack of an effective high-throughput screening (HTS) method. Here, we devise a catalase-deficient <i>Escherichia coli</i> host strain and report an HTS approach based on colorimetric detection of H<sub>2</sub>O<sub>2</sub>-consumption activity of FADCs. Directed evolution enabled by this method has led to effective identification for the first time of improved FADC variants for medium-chain 1-alkene production from both DNA shuffling and random mutagenesis libraries. Advantageously, this screening method can be extended to other enzymes that stoichiometrically utilize H<sub>2</sub>O<sub>2</sub> as co-substrate.

scholarly journals Development of a Novel High-Throughput Screen and Identification of Small-Molecule Inhibitors of the Gα–RGS17 Protein–Protein Interaction Using AlphaScreen

2011 ◽  
Vol 16 (8) ◽  
pp. 869-877 ◽  
Author(s):  
Duncan I. Mackie ◽  
David L. Roman
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
Protein Interaction ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Screening Method ◽  
Fold Increase ◽  
Rgs Proteins ◽  
High Throughput Screen ◽  
Protein Protein Interaction

In this study, the authors used AlphaScreen technology to develop a high-throughput screening method for interrogating small-molecule libraries for inhibitors of the Gαo–RGS17 interaction. RGS17 is implicated in the growth, proliferation, metastasis, and the migration of prostate and lung cancers. RGS17 is upregulated in lung and prostate tumors up to a 13-fold increase over patient-matched normal tissues. Studies show RGS17 knockdown inhibits colony formation and decreases tumorigenesis in nude mice. The screen in this study uses a measurement of the Gαo–RGS17 protein–protein interaction, with an excellent Z score exceeding 0.73, a signal-to-noise ratio >70, and a screening time of 1100 compounds per hour. The authors screened the NCI Diversity Set II and determined 35 initial hits, of which 16 were confirmed after screening against controls. The 16 compounds exhibited IC50 <10 µM in dose–response experiments. Four exhibited IC50 values <6 µM while inhibiting the Gαo–RGS17 interaction >50% when compared to a biotinylated glutathione-S-transferase control. This report describes the first high-throughput screen for RGS17 inhibitors, as well as a novel paradigm adaptable to many other RGS proteins, which are emerging as attractive drug targets for modulating G-protein-coupled receptor signaling.

scholarly journals A high-throughput screening method for evolving a demethylase enzyme with improved and new functionalities

2020 ◽  
Author(s):  
Yuru Wang ◽  
Christopher D Katanski ◽  
Christopher Watkins ◽  
Jessica N Pan ◽  
Qing Dai ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method ◽  
Targeted Mutagenesis ◽  
Rna Repair ◽  
Dna And Rna ◽  
Modified Dna ◽  
Dna Substrates ◽  
Trna Sequencing

Abstract AlkB is a DNA/RNA repair enzyme that removes base alkylations such as N1-methyladenosine (m1A) or N3-methylcytosine (m3C) from DNA and RNA. The AlkB enzyme has been used as a critical tool to facilitate tRNA sequencing and identification of mRNA modifications. As a tool, AlkB mutants with better reactivity and new functionalities are highly desired; however, previous identification of such AlkB mutants was based on the classical approach of targeted mutagenesis. Here, we introduce a high-throughput screening method to evaluate libraries of AlkB variants for demethylation activity on RNA and DNA substrates. This method is based on a fluorogenic RNA aptamer with an internal modified RNA/DNA residue which can block reverse transcription or introduce mutations leading to loss of fluorescence inherent in the cDNA product. Demethylation by an AlkB variant eliminates the blockage or mutation thereby restores the fluorescence signals. We applied our screening method to sites D135 and R210 in the Escherichia coli AlkB protein and identified a variant with improved activity beyond a previously known hyperactive mutant toward N1-methylguanosine (m1G) in RNA. We also applied our method to O6-methylguanosine (O6mG) modified DNA substrates and identified candidate AlkB variants with demethylating activity. Our study provides a high-throughput screening method for in vitro evolution of any demethylase enzyme.

scholarly journals A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A

2021 ◽  
Vol 22 (6) ◽  
pp. 3041
Author(s):  
Gheorghita Menghiu ◽  
Vasile Ostafe ◽  
Radivoje Prodanović ◽  
Rainer Fischer ◽  
Raluca Ostafe
Keyword(s):  
High Throughput ◽  
Cell Sorting ◽  
High Throughput Screening ◽  
Screening Method ◽  
Enzymatic Reaction ◽  
Hydrolytic Enzymes ◽  
Screening System ◽  
Fluorescence Activated Cell Sorting ◽  
Fungal Cell ◽  
Chitinase A

Chitinases catalyze the degradation of chitin, a polymer of N-acetylglucosamine found in crustacean shells, insect cuticles, and fungal cell walls. There is great interest in the development of improved chitinases to address the environmental burden of chitin waste from the food processing industry as well as the potential medical, agricultural, and industrial uses of partially deacetylated chitin (chitosan) and its products (chito-oligosaccharides). The depolymerization of chitin can be achieved using chemical and physical treatments, but an enzymatic process would be more environmentally friendly and more sustainable. However, chitinases are slow-acting enzymes, limiting their biotechnological exploitation, although this can be overcome by molecular evolution approaches to enhance the features required for specific applications. The two main goals of this study were the development of a high-throughput screening system for chitinase activity (which could be extrapolated to other hydrolytic enzymes), and the deployment of this new method to select improved chitinase variants. We therefore cloned and expressed the Bacillus licheniformis DSM8785 chitinase A (chiA) gene in Escherichia coli BL21 (DE3) cells and generated a mutant library by error-prone PCR. We then developed a screening method based on fluorescence-activated cell sorting (FACS) using the model substrate 4-methylumbelliferyl β-d-N,N′,N″-triacetyl chitotrioside to identify improved enzymes. We prevented cross-talk between emulsion compartments caused by the hydrophobicity of 4-methylumbelliferone, the fluorescent product of the enzymatic reaction, by incorporating cyclodextrins into the aqueous phases. We also addressed the toxicity of long-term chiA expression in E. coli by limiting the reaction time. We identified 12 mutants containing 2–8 mutations per gene resulting in up to twofold higher activity than wild-type ChiA.

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.

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