Development of a High-Content Screening Assay Panel to Accelerate Mechanism of Action Studies for Oncology Research

2012 ◽  
Vol 17 (8) ◽  
pp. 1005-1017 ◽  
Author(s):  
Danli L. Towne ◽  
Emily E. Nicholl ◽  
Kenneth M. Comess ◽  
Scott C. Galasinski ◽  
Philip J. Hajduk ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Mechanism Of Action ◽  
Large Scale ◽  
Morphological Changes ◽  
Single Cells ◽  
High Content Screening ◽  
Individual Compound ◽  
Screening Assay ◽  
Oncology Research ◽  
Protein Functions

Efficient elucidation of the biological mechanism of action of novel compounds remains a major bottleneck in the drug discovery process. To address this need in the area of oncology, we report the development of a multiparametric high-content screening assay panel at the level of single cells to dramatically accelerate understanding the mechanism of action of cell growth–inhibiting compounds on a large scale. Our approach is based on measuring 10 established end points associated with mitochondrial apoptosis, cell cycle disruption, DNA damage, and cellular morphological changes in the same experiment, across three multiparametric assays. The data from all of the measurements taken together are expected to help increase our current understanding of target protein functions, constrain the list of possible targets for compounds identified using phenotypic screens, and identify off-target effects. We have also developed novel data visualization and phenotypic classification approaches for detailed interpretation of individual compound effects and navigation of large collections of multiparametric cellular responses. We expect this general approach to be valuable for drug discovery across multiple therapeutic areas.

scholarly journals Development and pilot screen of novel high content assay for down regulators of expression of heterogenous nuclear ribonuclear protein H2

2020 ◽  
Author(s):  
Juan Diez ◽  
Sumitha Rajendrarao ◽  
Shadi A. Baajour ◽  
Praathibha Sripadhan ◽  
Timothy P. Spicer ◽  
...  
Keyword(s):  
Metastatic Melanoma ◽  
High Throughput Screening ◽  
Large Scale ◽  
Fluorescent Protein ◽  
High Content Screening ◽  
Screening Assay ◽  
Down Regulation ◽  
Metastatic Melanoma Cell ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein

ABSTRACTDespite recent advances in melanoma drug discovery, the average overall survival of patients with late stage metastatic melanoma is approximately 3 years, suggesting a need for new approaches and melanoma therapeutic targets. Previously we identified heterogeneous nuclear ribonucleoprotein H2 as a potential target of anti-melanoma compound 2155-14 (Palrasu et al, Cell Physiol Biochem 2019;53:656-86). In the present study, we endeavored to develop an assay to enable a high throughput screening campaign to identify drug-like molecules acting via down regulation of heterogeneous nuclear ribonucleoprotein H that can be used for melanoma therapy and research.ResultsWe established a cell-based platform using metastatic melanoma cell line WM266-4 expressing hnRNPH2 conjugated with green fluorescent protein to enable assay development and screening. High Content Screening assay was developed and validated in 384 well plate format, followed by miniaturization to 1,536 well plate format. All plate-based QC parameters were acceptable: %CV = 6.7±0.3, S/B = 21±2.1, Z’ = 0.75±0.04. Pilot screen of FDA-approved drug library (n=1,400 compounds) demonstrated hit rate of 0.5%. Two compounds demonstrated pharmacological response and were authenticated by western blot analysis.ConclusionsWe developed a highly robust HTS-amenable high content screening assay capable of monitoring down regulation of hnRNPH2. This assay is thus capable of identifying authentic down regulators of hnRNPH1 and 2 in a large compound collection and, therefore, is amenable to a large-scale screening effort.

A novel multi-parametric high content screening assay in ciPTEC-OAT1 to predict drug-induced nephrotoxicity during drug discovery

2018 ◽  
Vol 92 (10) ◽  
pp. 3175-3190 ◽  
Author(s):  
Anna-Karin Sjögren ◽  
Katarina Breitholtz ◽  
Ernst Ahlberg ◽  
Lucas Milton ◽  
Malin Forsgard ◽  
...  
Keyword(s):  
Drug Discovery ◽  
High Content Screening ◽  
Screening Assay ◽  
Drug Induced

scholarly journals High-Content Analysis-Based Sensitivity Prediction and Novel Therapeutics Screening for c-Met-Addicted Glioblastoma

Cancers ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 372
Author(s):  
Jeong-Woo Oh ◽  
Yun Jeong Oh ◽  
Suji Han ◽  
Nam-Gu Her ◽  
Do-Hyun Nam
Keyword(s):  
Content Analysis ◽  
Large Scale ◽  
Drug Sensitivity ◽  
Drug Repurposing ◽  
Genetic Alterations ◽  
High Content Screening ◽  
Precision Oncology ◽  
Novel Therapeutics ◽  
Oncology Research ◽  
High Content Analysis

(1) Background: Recent advances in precision oncology research rely on indicating specific genetic alterations associated with treatment sensitivity. Developing ex vivo systems to identify cancer patients who will respond to a specific drug remains important. (2) Methods: cells from 12 patients with glioblastoma were isolated, cultured, and subjected to high-content screening. Multi-parameter analyses assessed the c-Met level, cell viability, apoptosis, cell motility, and migration. A drug repurposing screen and large-scale drug sensitivity screening data across 59 cancer cell lines and patient-derived cells were obtained from 125 glioblastoma samples. (3) Results: High-content analysis of patient-derived cells provided robust and accurate drug responses to c-Met-targeted agents. Only the cells of one glioblastoma patient (PDC6) showed elevated c-Met level and high susceptibility to the c-Met inhibitors. Multi-parameter image analysis also reflected a decreased c-Met expression and reduced cell growth and motility by a c-Met-targeting antibody. In addition, a drug repurposing screen identified Abemaciclib as a distinct CDK4/6 inhibitor with a potent c-Met-inhibitory function. Consistent with this, we present large-scale drug sensitivity screening data showing that the Abemaciclib response correlates with the response to c-Met inhibitors. (4) Conclusions: Our study provides a new insight into high-content screening platforms supporting drug sensitivity prediction and novel therapeutics screening.

scholarly journals Development and Pilot Screen of Novel High Content Assay for Down Regulators of Expression of Heterogenous Nuclear Ribonuclear Protein H2

2021 ◽  
Vol 55 (3) ◽  
pp. 265-276
Keyword(s):  
Metastatic Melanoma ◽  
High Throughput Screening ◽  
Large Scale ◽  
Fluorescent Protein ◽  
High Content Screening ◽  
Screening Assay ◽  
Down Regulation ◽  
Metastatic Melanoma Cell ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein

BACKGROUND/AIMS: Despite recent advances in melanoma drug discovery, the average overall survival of patients with late-stage metastatic melanoma is approximately 3 years, suggesting a need for new approaches and melanoma therapeutic targets. Previously we identified heterogeneous nuclear ribonucleoprotein H2 as a potential target of anti-melanoma compound 2155-14 (Palrasu et al., Cell Physiol Biochem 2019;53:656-686). In the present study, we endeavored to develop an assay to enable a high throughput screening campaign to identify drug-like molecules acting via down regulation of heterogeneous nuclear ribonucleoprotein H2 that can be used for melanoma therapy and research. METHODS: We established a cell-based platform using metastatic melanoma cell line WM266-4 expressing hnRNPH2 conjugated with green fluorescent protein to enable assay development and screening. High Content Screening assay was developed and validated in 384 well plate format, followed by miniaturization to 1,536 well plate format. RESULTS: All plate-based QC parameters were acceptable: %CV = 6.7±0.3, S/B = 21±2.1, Z' = 0.75±0.04. Pilot screen of FDA-approved drug library (n=1,400 compounds) demonstrated hit rate of 0.5%. Two compounds demonstrated pharmacological response and were authenticated by western blot analysis. CONCLUSION: We developed a highly robust HTS-amenable high content screening assay capable of monitoring down regulation of hnRNPH2. This assay is thus capable of identifying authentic down regulators of hnRNPH1 and 2 in a large compound collection and, therefore, is amenable to a large-scale screening effort.

scholarly journals Roadmap for the use of base editors to decipher drug mechanism of action

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0257537
Author(s):  
Estel Aparicio-Prat ◽  
Dong Yan ◽  
Marco Mariotti ◽  
Michael Bassik ◽  
Gaelen Hess ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Mechanism Of Action ◽  
Large Scale ◽  
Reporter System ◽  
Experimental Conditions ◽  
Drug Mechanism ◽  
Β2 Adrenergic Receptor ◽  
Set Up ◽  
G Protein Coupled ◽  
Early Drug

CRISPR base editors are powerful tools for large-scale mutagenesis studies. This kind of approach can elucidate the mechanism of action of compounds, a key process in drug discovery. Here, we explore the utility of base editors in an early drug discovery context focusing on G-protein coupled receptors. A pooled mutagenesis screening framework was set up based on a modified version of the CRISPR-X base editor system. We determine optimized experimental conditions for mutagenesis where sgRNAs are delivered by cell transfection or viral infection over extended time periods (>14 days), resulting in high mutagenesis produced in a short region located at -4/+8 nucleotides with respect to the sgRNA match. The β2 Adrenergic Receptor (B2AR) was targeted in this way employing a 6xCRE-mCherry reporter system to monitor its response to isoproterenol. The results of our screening indicate that residue 184 of B2AR is crucial for its activation. Based on our experience, we outline the crucial points to consider when designing and performing CRISPR-based pooled mutagenesis screening, including the typical technical hurdles encountered when studying compound pharmacology.

scholarly journals Roadmap for the use of Base Editors to Decipher Drug Mechanism of Action

2020 ◽  
Author(s):  
Estel Aparicio Prat ◽  
Dong Yan ◽  
Marco Mariotti ◽  
Michael Bassik ◽  
Gaelen Hess ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Mechanism Of Action ◽  
Large Scale ◽  
Reporter System ◽  
Experimental Conditions ◽  
Base Editing ◽  
Drug Mechanism ◽  
Set Up ◽  
G Protein Coupled ◽  
Early Drug

Abstract Background: CRISPR base editors are powerful tools for large-scale mutagenesis studies. This kind of approach can elucidate the mechanism of action of compounds, a key process in drug discovery. Here, we explore the utility of base editors in an early drug discovery context, and we focus on G-protein coupled receptors.Results: We set up a pooled mutagenesis screening framework based on a modified version of the CRISPR-X base editor system. We determine optimized experimental conditions for mutagenesis where sgRNAs are delivered by cell transfection or viral infection over extended time periods (>14 days), resulting in high mutagenesis produced in a short region located at -4/+8 nucleotides with respect to the sgRNA match. We thus target the Beta 2 Adrenergic Receptor (B2AR) and employ a 6xCRE-mCherry reporter system to monitor its activity. The results of our screening indicate that residue 184 of B2AR is crucial for its activation. Based on our experience, we then outline the crucial points to consider when designing and performing CRISPR-based pooled mutagenesis screening, including the typical technical hurdles encountered when studying compound pharmacology. Conclusions: The base editing technology has a great potential to help deciphering the mechanism of action of drugs, and it is a very powerful tool in drug discovery. Here we show an application of pooled mutagenesis screening to study B2AR, and we provide a roadmap for successfully applying this approach to other target proteins.

Large-Scale Assessment of Binding Free Energy Calculations in Active Drug Discovery Projects

2020 ◽  
Author(s):  
Christina Schindler ◽  
Hannah Baumann ◽  
Andreas Blum ◽  
Dietrich Böse ◽  
Hans-Peter Buchstaller ◽  
...  
Keyword(s):  
Free Energy ◽  
Drug Discovery ◽  
Large Scale ◽  
Active Drug ◽  
Binding Free Energy ◽  
Free Energy Calculations ◽  
Energy Calculation ◽  
Large Scale Assessment ◽  
New Public ◽  
Binding Free Energy Calculations

Here we present an evaluation of the binding affinity prediction accuracy of the free energy calculation method FEP+ on internal active drug discovery projects and on a large new public benchmark set.<br>

Reaction-based Enumeration, Active Learning, and Free Energy Calculations to Rapidly Explore Synthetically Tractable Chemical Space and Optimize Potency of Cyclin Dependent Kinase 2 Inhibitors

2019 ◽  
Author(s):  
Kyle Konze ◽  
Pieter Bos ◽  
Markus Dahlgren ◽  
Karl Leswing ◽  
Ivan Tubert-Brohman ◽  
...  
Keyword(s):  
Free Energy ◽  
Drug Discovery ◽  
Active Learning ◽  
Large Scale ◽  
Chemical Space ◽  
Population Based ◽  
Free Energy Calculations ◽  
Computational Technique ◽  
Cyclin Dependent Kinase ◽  
Energy Calculations

We report a new computational technique, PathFinder, that uses retrosynthetic analysis followed by combinatorial synthesis to generate novel compounds in synthetically accessible chemical space. Coupling PathFinder with active learning and cloud-based free energy calculations allows for large-scale potency predictions of compounds on a timescale that impacts drug discovery. The process is further accelerated by using a combination of population-based statistics and active learning techniques. Using this approach, we rapidly optimized R-groups and core hops for inhibitors of cyclin-dependent kinase 2. We explored greater than 300 thousand ideas and identified 35 ligands with diverse commercially available R-groups and a predicted IC<sub>50</sub> < 100 nM, and four unique cores with a predicted IC<sub>50</sub> < 100 nM. The rapid turnaround time, and scale of chemical exploration, suggests that this is a useful approach to accelerate the discovery of novel chemical matter in drug discovery campaigns.

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