High-Throughput Mechanism of Inhibition

2021 ◽  
Vol 26 (2) ◽  
pp. 248-256
Author(s):  
Gareth Davies ◽  
Hannah Semple ◽  
Megan McCandless ◽  
Jonathan Cairns ◽  
Geoffrey A. Holdgate
Keyword(s):  
High Throughput ◽  
Substrate Concentration ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Significant Proportion ◽  
Mechanism Of Inhibition ◽  
The Common ◽  
High Throughput Screens ◽  
The Impact ◽  
Druggable Genome

Enzymes represent a significant proportion of the druggable genome and constitute a rich source of drug targets. Delivery of a successful program for developing a modulator of enzyme activity requires an understanding of the enzyme’s mechanism and the mode of interaction of compounds. This allows an understanding of how physiological conditions in disease-relevant cells will affect inhibitor potency. As a result, there is increasing interest in evaluating hit compounds from high-throughput screens to determine their mode of interaction with the target. This work revisits the common inhibition modalities and illustrates the impact of substrate concentration relative to Km upon the pattern of changes in IC50 that are expected for increasing substrate concentration. It proposes a new, high-throughput approach for assessing mode of inhibition, incorporating analyses based on a minimal descriptive model, to deliver a workflow that allows rapid and earlier compound classification immediately after high-throughput screening.

scholarly journals Development of a High-Throughput Screening Method for LIM Kinase 1 Using a Luciferase-Based Assay of ATP Consumption

2011 ◽  
Vol 17 (4) ◽  
pp. 460-468 ◽  
Author(s):  
Mokdad Mezna ◽  
Ai Ching Wong ◽  
Margaret Ainger ◽  
Rebecca W. Scott ◽  
Tim Hammonds ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Kinase Inhibitors ◽  
Screening Method ◽  
Human Diseases ◽  
Luciferase Assay ◽  
Lim Kinase ◽  
Binding Pockets ◽  
High Throughput Screens

Kinases are attractive drug targets because of the central roles they play in signal transduction pathways and human diseases. Their well-formed adenosine triphosphate (ATP)–binding pockets make ideal targets for small-molecule inhibitors. For drug discovery purposes, many peptide-based kinase assays have been developed that measure substrate phosphorylation using fluorescence-based readouts. However, for some kinases these assays may not be appropriate. In the case of the LIM kinases (LIMK), an inability to phosphorylate peptide substrates resulted in previous high-throughput screens (HTS) using radioactive labeling of recombinant cofilin protein as the readout. We describe the development of an HTS-compatible assay that measures relative ATP levels using luciferase-generated luminescence as a function of LIMK activity. The assay was inexpensive to perform, and proof-of-principle screening of kinase inhibitors demonstrated that compound potency against LIMK could be determined; ultimately, the assay was used for successful prosecution of automated HTS. Following HTS, the secondary assay format was changed to obtain more accurate measures of potency and mechanism of action using more complex (and expensive) assays. The luciferase assay nonetheless provides an inexpensive and reliable primary assay for HTS that allowed for the identification of LIMK inhibitors to initiate discovery programs for the eventual treatment of human diseases.

scholarly journals Predicting perturbation patterns from the topology of biological networks

2018 ◽  
Author(s):  
Marc Santolini ◽  
Albert-László Barabási
Keyword(s):  
Kinetic Parameters ◽  
High Throughput ◽  
Drug Targets ◽  
Rapid Development ◽  
Kinetic Constant ◽  
Significant Proportion ◽  
Biochemical Networks ◽  
Human Interactome ◽  
Knock Out ◽  
The Impact

AbstractHigh-throughput technologies, offering unprecedented wealth of quantitative data underlying the makeup of living systems, are changing biology. Notably, the systematic mapping of the relationships between biochemical entities has fueled the rapid development of network biology, offering a suitable framework to describe disease phenotypes and predict potential drug targets. Yet, our ability to develop accurate dynamical models remains limited, due in part to the limited knowledge of the kinetic parameters underlying these interactions. Here, we explore the degree to which we can make reasonably accurate predictions in the absence of the kinetic parameters. We find that simple dynamically agnostic models are sufficient to recover the strength and sign of the biochemical perturbation patterns observed in 87 biological models for which the underlying kinetics is known. Surprisingly, a simple distance-based model achieves 65% accuracy. We show that this predictive power is robust to topological and kinetic parameters perturbations, and we identify key network properties that can increase up to 80% the recovery rate of the true perturbation patterns. We validate our approach using experimental data on the chemotactic pathway in bacteria, finding that a network model of perturbation spreading predicts with ~80% accuracy the directionality of gene expression and phenotype changes in knock-out and overproduction experiments. These findings show that the steady advances in mapping out the topology of biochemical interaction networks opens avenues for accurate perturbation spread modeling, with direct implications for medicine and drug development.Significance statementThe development of high-throughput technologies has allowed to map a significant proportion of interactions between biochemical entities in the cell. However, it is unclear how much information is lost given the lack of measurements on the kinetic parameters governing the dynamics of these interactions. Using biochemical networks with experimentally measured kinetic parameters, we show that a knowledge of the network topology offers 65% to 80% accuracy in predicting the impact of perturbation patterns. In other words, we can use the increasingly accurate topological models to approximate perturbation patterns, bypassing expensive kinetic constant measurement. These results could open new avenues in modeling drug action, and in identifying drug targets relying on the human interactome only.

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.

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 Cytotoxic Profiling of Annotated and Diverse Chemical Libraries Using Quantitative High-Throughput Screening

2019 ◽  
Vol 25 (1) ◽  
pp. 9-20 ◽  
Author(s):  
Olivia W. Lee ◽  
Shelley Austin ◽  
Madison Gamma ◽  
Dorian M. Cheff ◽  
Tobie D. Lee ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
High Throughput ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Large Scale ◽  
Early Stage ◽  
Cancer Cell Line ◽  
Hek 293 ◽  
Cytotoxic Compounds

Cell-based phenotypic screening is a commonly used approach to discover biological pathways, novel drug targets, chemical probes, and high-quality hit-to-lead molecules. Many hits identified from high-throughput screening campaigns are ruled out through a series of follow-up potency, selectivity/specificity, and cytotoxicity assays. Prioritization of molecules with little or no cytotoxicity for downstream evaluation can influence the future direction of projects, so cytotoxicity profiling of screening libraries at an early stage is essential for increasing the likelihood of candidate success. In this study, we assessed the cell-based cytotoxicity of nearly 10,000 compounds in the National Institutes of Health, National Center for Advancing Translational Sciences annotated libraries and more than 100,000 compounds in a diversity library against four normal cell lines (HEK 293, NIH 3T3, CRL-7250, and HaCat) and one cancer cell line (KB 3-1, a HeLa subline). This large-scale library profiling was analyzed for overall screening outcomes, hit rates, pan-activity, and selectivity. For the annotated library, we also examined the primary targets and mechanistic pathways regularly associated with cell death. To our knowledge, this is the first study to use high-throughput screening to profile a large screening collection (>100,000 compounds) for cytotoxicity in both normal and cancer cell lines. The results generated here constitute a valuable resource for the scientific community and provide insight into the extent of cytotoxic compounds in screening libraries, allowing for the identification and avoidance of compounds with cytotoxicity during high-throughput screening campaigns.

scholarly journals Platforms for High-Throughput Screening and Force Measurements on Fungi and Oomycetes

Micromachines ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 639
Author(s):  
Yiling Sun ◽  
Ayelen Tayagui ◽  
Sarah Sale ◽  
Debolina Sarkar ◽  
Volker Nock ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Control Strategies ◽  
Pathogenic Fungi ◽  
Antifungal Drugs ◽  
Plant Diseases ◽  
Force Measurements ◽  
Technical Developments ◽  
Alternative Drug

Pathogenic fungi and oomycetes give rise to a significant number of animal and plant diseases. While the spread of these pathogenic microorganisms is increasing globally, emerging resistance to antifungal drugs is making associated diseases more difficult to treat. High-throughput screening (HTS) and new developments in lab-on-a-chip (LOC) platforms promise to aid the discovery of urgently required new control strategies and anti-fungal/oomycete drugs. In this review, we summarize existing HTS and emergent LOC approaches in the context of infection strategies and invasive growth exhibited by these microorganisms. To aid this, we introduce key biological aspects and review existing HTS platforms based on both conventional and LOC techniques. We then provide an in-depth discussion of more specialized LOC platforms for force measurements on hyphae and to study electro- and chemotaxis in spores, approaches which have the potential to aid the discovery of alternative drug targets on future HTS platforms. Finally, we conclude with a brief discussion of the technical developments required to improve the uptake of these platforms into the general laboratory environment.

A systematic approach to prioritize drug targets using machine learning, a molecular descriptor-based classification model, and high-throughput screening of plant derived molecules: a case study in oral cancer

2015 ◽  
Vol 11 (12) ◽  
pp. 3362-3377 ◽  
Author(s):  
Vinay Randhawa ◽  
Anil Kumar Singh ◽  
Vishal Acharya
Keyword(s):  
Machine Learning ◽  
Oral Cancer ◽  
High Throughput ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Systematic Approach ◽  
Molecular Descriptor ◽  
Classification Model

Network-based and cheminformatics approaches identify novel lead molecules forCXCR4, a key gene prioritized in oral cancer.

scholarly journals Identification of several high-risk HPV inhibitors and drug targets with a novel high-throughput screening assay

2017 ◽  
Vol 13 (2) ◽  
pp. e1006168 ◽  
Author(s):  
Mart Toots ◽  
Mart Ustav ◽  
Andres Männik ◽  
Karl Mumm ◽  
Kaido Tämm ◽  
...  
Keyword(s):  
High Risk ◽  
High Throughput ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
High Risk Hpv

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.

scholarly journals Stability of Screening Compounds in Wet DMSO

2008 ◽  
Vol 13 (10) ◽  
pp. 999-1006 ◽  
Author(s):  
Caroline Engeloch ◽  
Ulrich Schopfer ◽  
Ingo Muckenschnabel ◽  
Francois Le Goff ◽  
Hervé Mees ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Medicinal Chemistry ◽  
Storage Conditions ◽  
Pragmatic Approach ◽  
Analytical Quality ◽  
The Past ◽  
Effect Of Water ◽  
The Impact

The impact of storage conditions on compound stability and compound solubility has been debated intensely over the past 5 years. At Novartis, the authors decided to opt for a storage concept that can be considered controversial because they are using a DMSO/water (90/10) mixture as standard solvent. To assess the effect of water in DMSO stocks on compound stability, the authors monitored the purity of a subset of 1404 compounds from ongoing medicinal chemistry projects over several months. The study demonstrated that 85% of the compounds were stable in wet DMSO over a 2-year period at 4 °C. This result validates the storage concept developed at Novartis as a pragmatic approach that takes advantage of the benefits of DMSO/water mixtures while mediating the disadvantages. In addition, the authors describe how purity data collected over the course of the chemical validation of high-throughput screening actives are used to improve the analytical quality of the Novartis screening deck. ( Journal of Biomolecular Screening 2008:999-1006)

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