scholarly journals The trials and tribulations of a robotic screening core

1995 ◽  
Vol 17 (2) ◽  
pp. 55-58 ◽  
Author(s):  
John Babiak ◽  
Brian Lucotch ◽  
Anthony Russo ◽  
Linda Heydt ◽  
Sharon Williams ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Customer Relations ◽  
Chemical Compounds ◽  
Screen Design ◽  
Therapeutic Benefits ◽  
Primary Means ◽  
Effective Use ◽  
High Throughput Screens ◽  
Time Frames

It is well recognized within the pharmaceutical industry that high throughput screening is a valuable and rapid tool to identify novel chemical compounds that may lead to tomorrow's drugs. High throughput screening involves testing as many chemical compounds as quickly as possible against a defined molecular or cellular ‘target’ (for example an enzyme) in the hope that interacting compounds may provide significant therapeutic benefits.At Wyeth-Ayerst Research, a Robotics and Automation Research Core Group has been established which serves as the in-house resource for high throughput screening. The robotics group has three missions: (1) develop and perform high throughput screens for customers in all therapeutic departments in the company; (2) educate customers in issues related to screen design; and (3) help customers to bring automated workstations into their laboratories. The mission, therefore, requires the effective use of automation, as well as building a strong collaboration with customers.The challenges that have been faced fall into two categories: technology limiting and customer relations. Technological challenges arise because it is necessary to develop and implement assays with very different formats and biochemical endpoints within extremely shortened time frames. The primary means to meet these challenges is with flexible robotics and flexible people. Challenges in the area of customer relations include setting realistic expectations, maintaining a sense of collaboration (and not merely service), educating investigators as to how to deal with the huge amount of data generated and seeking feedback. Effective and frequent communication, and an awareness of each individual's perspective, are essential to provide the most appropriate service.

High-Throughput Screening to Identify Small Molecules That Selectively Inhibit APOL1 Protein Level in Podocytes

2021 ◽  
pp. 247255522110262
Author(s):  
Jonathan Choy ◽  
Yanqing Kan ◽  
Steve Cifelli ◽  
Josephine Johnson ◽  
Michelle Chen ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Strategy ◽  
Time Resolved ◽  
Protein Levels ◽  
Increased Risk ◽  
Compound Screening ◽  
High Throughput Screens ◽  
Screening Library

High-throughput phenotypic screening is a key driver for the identification of novel chemical matter in drug discovery for challenging targets, especially for those with an unclear mechanism of pathology. For toxic or gain-of-function proteins, small-molecule suppressors are a targeting/therapeutic strategy that has been successfully applied. As with other high-throughput screens, the screening strategy and proper assays are critical for successfully identifying selective suppressors of the target of interest. We executed a small-molecule suppressor screen to identify compounds that specifically reduce apolipoprotein L1 (APOL1) protein levels, a genetically validated target associated with increased risk of chronic kidney disease. To enable this study, we developed homogeneous time-resolved fluorescence (HTRF) assays to measure intracellular APOL1 and apolipoprotein L2 (APOL2) protein levels and miniaturized them to 1536-well format. The APOL1 HTRF assay served as the primary assay, and the APOL2 and a commercially available p53 HTRF assay were applied as counterscreens. Cell viability was also measured with CellTiter-Glo to assess the cytotoxicity of compounds. From a 310,000-compound screening library, we identified 1490 confirmed primary hits with 12 different profiles. One hundred fifty-three hits selectively reduced APOL1 in 786-O, a renal cell adenocarcinoma cell line. Thirty-one of these selective suppressors also reduced APOL1 levels in conditionally immortalized human podocytes. The activity and specificity of seven resynthesized compounds were validated in both 786-O and podocytes.

scholarly journals An HTRF based high-throughput screening for discovering chemical compounds that inhibit the interaction between Trypanosoma brucei Pex5p and Pex14p

2016 ◽  
Vol 6 ◽  
pp. 260-265 ◽  
Author(s):  
Yuichi Watanabe ◽  
Kosuke Kawaguchi ◽  
Syuken Saito ◽  
Takayoshi Okabe ◽  
Kiyoaki Yonesu ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
High Throughput ◽  
High Throughput Screening ◽  
Chemical Compounds

scholarly journals High-Throughput Screening for Human Galactokinase Inhibitors

2008 ◽  
Vol 13 (5) ◽  
pp. 415-423 ◽  
Author(s):  
Klaas J. Wierenga ◽  
Kent Lai ◽  
Peter Buchwald ◽  
Manshu Tang
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Expression System ◽  
Chemical Compounds ◽  
Average Concentration ◽  
Effective Therapy ◽  
Experimental Conditions ◽  
Classic Galactosemia ◽  
Neonatal Lethality

Inherited deficiency of galactose-1-phosphate uridyltransferase (GALT) can result in a potentially lethal disorder called classic galactosemia. Although the neonatal lethality associated with this disease can be prevented through early diagnosis and a galactose-restricted diet, the lack of effective therapy continues to have consequences: developmental delay, neurological disorders, and premature ovarian failure are common sequelae in childhood and adulthood. Several lines of evidence indicate that an elevated level of galactose-1-phosphate (gal-1-p), the product of galactokinase (GALK), is a major, if not sole, pathogenic mechanism in patients with classic galactosemia. The authors hypothesize that elimination of gal-1-p production by inhibiting GALK will relieve GALT-deficient cells from galactose toxicity. To test this hypothesis, they obtained human GALK using a bacterial expression system. They developed a robust, miniaturized, high-throughput GALK assay (Z′ factor = 0.91) and used this assay to screen against libraries composed of 50,000 chemical compounds with diverse structural scaffolds. They selected 150 compounds that, at an average concentration of 33.3 µM, inhibited GALK activity in vitro more than 86.5% and with a reproducibility score of at least 0.7 for a confirmatory screen under identical experimental conditions. Of these 150 compounds, 34 were chosen for further characterization. Preliminary results indicated that these 34 compounds have potential to serve as leads to the development of more effective therapy of classic galactosemia. ( Journal of Biomolecular Screening 2008:415-423)

Transforming Your Robotics Into an Infrastructure for the Future

1997 ◽  
Vol 2 (3) ◽  
pp. 139-143 ◽  
Author(s):  
John Babiak
Keyword(s):  
Data Management ◽  
High Throughput ◽  
High Throughput Screening ◽  
Core Competencies ◽  
The Other ◽  
Exploratory Research ◽  
Screening Process ◽  
Screen Design ◽  
Bar Codes ◽  
Computer Based

The four infrastructural elements for effective high throughput screening are Sample Sourcing, Screen Design, Robotic Hardware, and Data Management. These must all work together to achieve a balance between flexibility and productivity. Sample Sourcing can enhance the high throughput screening process when bar codes are used in a consistent and sensible manner which takes into account the way people think and work. Exploratory research that leads to core competencies in Screen Design can vastly streamline the development and implementation of a high throughput screen by providing scientist customers with specific guidelines which capitalize on your robotic strengths. Robotic Hardware architecture should be open to extension, computer-based, reliable, easy to use, and modified on an ongoing basis. Data Management needs to be pervasive and tie together the other components of the infrastructure. The essential ingredients, though, are motivated and well-trained people who will transform the tools of robotics and automation into the infrastructure needed for effective drug discovery.

scholarly journals High-Throughput 5’ UTR Engineering for Enhanced Protein Production in Non-Viral Gene Therapies

2020 ◽  
Author(s):  
Jicong Cao ◽  
Eva Maria Novoa ◽  
Zhizhuo Zhang ◽  
William C.W. Chen ◽  
Dianbo Liu ◽  
...  
Keyword(s):  
Protein Expression ◽  
High Throughput ◽  
High Throughput Screening ◽  
Viral Gene ◽  
Cmv Promoter ◽  
Position Effects ◽  
Gene Therapies ◽  
Integration Strategy ◽  
Combinatorial Assembly ◽  
High Throughput Screens

ABSTRACTDespite significant clinical progress in cell and gene therapies, maximizing protein expression in order to enhance potency remains a major challenge. One approach to increase protein expression is by optimizing translation through the engineering of 5’ untranslated regions (5’ UTRs). Here, we developed a high-throughput strategy to design, screen, and optimize novel 5’UTRs that enhance protein expression from a strong human cytomegalovirus (CMV) promoter. We first identified naturally occurring 5’ UTRs with high translation efficiencies and used this information with in silico genetic algorithms to generate synthetic 5’ UTRs. A total of ∼12,000 5’ UTRs were then screened using a recombinase-mediated integration strategy that greatly enhances the sensitivity of high-throughput screens by eliminating copy number and position effects that limit lentiviral approaches. Using this approach, we identified three synthetic 5’ UTRs that outperformed commonly used non-viral gene therapy plasmids in expressing protein payloads. Furthermore, combinatorial assembly of these 5’ UTRs enabled even higher protein expression than obtained with each individual 5’ UTR. In summary, we demonstrate that high-throughput screening of 5’ UTR libraries with recombinase-mediated integration can identify genetic elements that enhance protein expression, which should have numerous applications for engineered cell and gene therapies.

scholarly journals Identifying Anti-prion Chemical Compounds Using a Newly Established Yeast High-Throughput Screening System

2019 ◽  
Vol 26 (12) ◽  
pp. 1664-1680.e4 ◽  
Author(s):  
Zhiqiang Du ◽  
Stephanie Valtierra ◽  
Luzivette Robles Cardona ◽  
Sara Fernandez Dunne ◽  
Chi-Hao Luan ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Chemical Compounds ◽  
Screening System

High-Throughput Screening for Small-Molecule Adiponectin Secretion Modulators

2011 ◽  
Vol 16 (6) ◽  
pp. 628-636 ◽  
Author(s):  
Kyosuke Hino ◽  
Hidetaka Nagata ◽  
Manabu Shimonishi ◽  
Motoharu Ido
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor ◽  
Drug Candidates ◽  
Insulin Resistant ◽  
Therapeutic Benefits ◽  
Pparγ Agonists ◽  
Agonistic Activity

Adiponectin is an adipokine secreted by adipocytes and plays a role in the suppression of metabolic disorders that can result in type 2 diabetes, obesity, and atherosclerosis. Several studies have shown that upregulation of adiponectin has a number of therapeutic benefits. Although peroxisome proliferator-activated receptor γ (PPARγ) agonists are known to increase adiponectin secretion both in cultured adipocytes and humans, they have several side effects, such as weight gain, congestive heart failure, and edema. Therefore, adiponectin secretion modulators that do not possess PPARγ agonistic activity seem to promising for a number of conditions. Here, the authors report on the development of a reporter-based high-throughput screening (HTS) assay using insulin-resistant-mimic 3T3-L1 adipocytes for discovery of adiponectin secretion modulators. They screened a library of approximately 100 000 small-molecule compounds using this model, performed several follow-up screens, and identified six hit compounds that increase adiponectin secretion without having PPARγ agonistic activity. These compounds may be useful drug candidates for diabetes, obesity, atherosclerosis, and other metabolic syndromes. This HTS assay might be applicable to screening for other adipokine modulators that can be useful for the treatment of other conditions.

scholarly journals RefCell: Multi-dimensional analysis of image-based high-throughput screens based on ‘typical cells’

2018 ◽  
Author(s):  
Yang Shen ◽  
Nard Kubben ◽  
Julián Candia ◽  
Alexandre V. Morozov ◽  
Tom Misteli ◽  
...  
Keyword(s):  
Dimensional Analysis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Premature Aging ◽  
Cellular Heterogeneity ◽  
High Level ◽  
High Throughput Screens ◽  
High Throughput Imaging

AbstractBackgroundImage-based high-throughput screening (HTS) reveals a high level of heterogeneity in single cells and multiple cellular states may be observed within a single population. Cutting-edge high-dimensional analysis methods are successful in characterizing cellular heterogeneity, but they suffer from the “curse of dimensionality” and non-standardized outputs.ResultsHere we introduce RefCell, a multi-dimensional analysis pipeline for image-based HTS that reproducibly captures cells with typical combinations of features in reference states, and uses these “typical cells” as a reference for classification and weighting of metrics. RefCell quantitatively assesses the heterogeneous deviations from typical behavior for each analyzed perturbation or sample.ConclusionsWe apply RefCell to the analysis of data from a high-throughput imaging screen of a library of 320 ubiquitin protein targeted siRNAs selected to gain insights into the mechanisms of premature aging (progeria). RefCell yields results comparable to a more complex clustering based single cell analysis method, which both reveal more potential hits than conventional average based analysis.

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