A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays

1999 ◽  
Vol 4 (2) ◽  
pp. 67-73 ◽  
Author(s):  
Ji-Hu Zhang ◽  
Thomas D. Y. Chung ◽  
Kevin R. Oldenburg
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Dynamic Range ◽  
Statistical Parameter ◽  
Assay Optimization ◽  
Screening Assays ◽  
Signal Dynamic Range ◽  
Hit Identification ◽  
Data Variation

The ability to identify active compounds ("hits") from large chemical libraries accurately and rapidly has been the ultimate goal in developing high-throughput screening (HTS) assays. The ability to identify hits from a particular HTS assay depends largely on the suitability or quality of the assay used in the screening. The criteria or parameters for evaluating the "suitability" of an HTS assay for hit identification are not well defined and hence it still remains difficult to compare the quality of assays directly. In this report, a screening window coefficient, called "Z- factor," is defined. This coefficient is reflective of both the assay signal dynamic range and the data variation associated with the signal measurements, and therefore is suitable for assay quality assessment. The Z-factor is a dimensionless, simple statistical characteristic for each HTS assay. The Z-factor provides a useful tool for comparison and evaluation of the quality of assays, and can be utilized in assay optimization and validation.

Development and Application of Activity-based Fluorescent Probes for High-Throughput Screening

2021 ◽  
Vol 28 ◽  
Author(s):  
Juan Cheng ◽  
Xin Li
Keyword(s):  
Drug Development ◽  
High Throughput ◽  
Fluorescent Probes ◽  
Analytical Methods ◽  
High Throughput Screening ◽  
High Sensitivity ◽  
Rapid Identification ◽  
Screening Assays ◽  
Hit Identification ◽  
Working Mechanisms

: High-throughput screening facilitates the rapid identification of novel hit compounds; however, it remains challenging to design effective high-throughput assays, partially due to the difficulty of achieving sensitivity in the assay techniques. Among the various analytical methods that are used, fluorescence-based assays dominate owing to their high sensitivity and ease of operation. Recent advances in activity-based sensing/imaging have further expanded the availability of fluorescent probes as monitors for high-throughput screening of result outputs. In this study, we have reviewed various activity-based fluorescent probes used in high-throughput screening assays, emphasizing their structure-related working mechanisms. Moreover, we have explored the possibility of the development of additional and better probes to boost hit identification and drug development against various targets.

scholarly journals Action plan for hit identification (APHID): KAT6A as a case study

2020 ◽  
Vol 12 (5) ◽  
pp. 423-437 ◽  
Author(s):  
Xiangyan Yi ◽  
Lian Xue ◽  
Tim Thomas ◽  
Jonathan B Baell
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Action Plan ◽  
Hit Identification

Here, we describe our action plan for hit identification (APHID) that guides the process of hit triage, with elimination of less tractable hits and retention of more tractable hits. We exemplify the process with reference to our high-throughput screening (HTS) campaign against the enzyme, KAT6A, that resulted in successful identification of a tractable hit. We hope that APHID could serve as a useful, concise and digestible guide for those involved in HTS and hit triage, especially those that are relatively new to this exciting and continually evolving technology.

scholarly journals Design of High-Throughput Screening Assays and Identification of a SUMO1-Specific Small Molecule Chemotype Targeting the SUMO-Interacting Motif-Binding Surface

2015 ◽  
Vol 17 (4) ◽  
pp. 239-246 ◽  
Author(s):  
Aileen Y. Alontaga ◽  
Yifei Li ◽  
Chih-Hong Chen ◽  
Chen-Ting Ma ◽  
Siobhan Malany ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Binding Surface ◽  
Screening Assays

scholarly journals Development of high throughput screening assays and pilot screen for inhibitors of metalloproteases meprin α and β

Biopolymers ◽  
2014 ◽  
Vol 102 (5) ◽  
pp. 396-406 ◽  
Author(s):  
Franck Madoux ◽  
Claudia Tredup ◽  
Timothy P. Spicer ◽  
Louis Scampavia ◽  
Peter S. Chase ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Assays

scholarly journals Rapid and Sensitive Quantification of Intracellular Glycyl-Sarcosine for Semi-High-Throughput Screening for Inhibitors of PEPT-1

Pharmaceutics ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1019
Author(s):  
Teresa von Linde ◽  
Gzona Bajraktari-Sylejmani ◽  
Walter E. Haefeli ◽  
Jürgen Burhenne ◽  
Johanna Weiss ◽  
...  
Keyword(s):  
Sample Preparation ◽  
High Throughput ◽  
High Throughput Screening ◽  
Dynamic Range ◽  
High Sensitivity ◽  
Peptide Bond ◽  
Peptide Transporter ◽  
Systemic Absorption ◽  
Screening Assay ◽  
Limit Of Quantification

The peptide transporter PEPT-1 (SLC15A1) plays a major role in nutritional supply with amino acids by mediating the intestinal influx of dipeptides and tripeptides generated during food digestion. Its role in the uptake of small bioactive peptides and various therapeutics makes it an important target for the investigation of the systemic absorption of small peptide-like active compounds and prodrug strategies of poorly absorbed therapeutics. The dipeptide glycyl-sarcosine (Gly-Sar), which comprises an N-methylated peptide bond that increases stability against enzymatic degradation, is widely utilized for studying PEPT-1-mediated transport. To support experiments on PEPT-1 inhibitor screening to identify potential substrates, we developed a highly sensitive Gly-Sar quantification assay for Caco-2 cell lysates with a dynamic range of 0.1 to 1000 ng/mL (lower limit of quantification 0.68 nM) in 50 µL of cell lysate. The assay was validated following the applicable recommendations for bioanalytic method validation of the FDA and EMA. Sample preparation and quantification were established in 96-well cell culture plates that were also used for the cellular uptake studies, resulting in a rapid and robust screening assay for PEPT-1 inhibitors. This sample preparation principle, combined with the high sensitivity of the UPLC-MS/MS quantification, is suitable for screening assays for PEPT-1 inhibitors and substrates in high-throughput formats and holds the potential for automation. Applicability was demonstrated by IC50 determinations of the known PEPT-1 inhibitor losartan, the known substrates glycyl-proline (Gly-Pro), and valaciclovir, the prodrug of aciclovir, which itself is no substrate of PEPT-1 and consequently showed no inhibition in our assay.

scholarly journals Clarifying intact 3D tissues on a microfluidic chip for high-throughput structural analysis

2016 ◽  
Vol 113 (52) ◽  
pp. 14915-14920 ◽  
Author(s):  
Yih Yang Chen ◽  
Pamuditha N. Silva ◽  
Abdullah Muhammad Syed ◽  
Shrey Sindhwani ◽  
Jonathan V. Rocheleau ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Three Dimensional ◽  
Microfluidic System ◽  
High Throughput Drug Screening ◽  
Image Analysis Algorithm ◽  
Screening Assays ◽  
On Chip ◽  
Tissue Models

On-chip imaging of intact three-dimensional tissues within microfluidic devices is fundamentally hindered by intratissue optical scattering, which impedes their use as tissue models for high-throughput screening assays. Here, we engineered a microfluidic system that preserves and converts tissues into optically transparent structures in less than 1 d, which is 20× faster than current passive clearing approaches. Accelerated clearing was achieved because the microfluidic system enhanced the exchange of interstitial fluids by 567-fold, which increased the rate of removal of optically scattering lipid molecules from the cross-linked tissue. Our enhanced clearing process allowed us to fluorescently image and map the segregation and compartmentalization of different cells during the formation of tumor spheroids, and to track the degradation of vasculature over time within extracted murine pancreatic islets in static culture, which may have implications on the efficacy of beta-cell transplantation treatments for type 1 diabetes. We further developed an image analysis algorithm that automates the analysis of the vasculature connectivity, volume, and cellular spatial distribution of the intact tissue. Our technique allows whole tissue analysis in microfluidic systems, and has implications in the development of organ-on-a-chip systems, high-throughput drug screening devices, and in regenerative medicine.

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