High-Density Miniaturized Thermal Shift Assays as a General Strategy for Drug Discovery

More general and universally applicable drug discovery assay technologies are needed in order to keep pace with the recent advances in combinatorial chemistry and genomics-based target generation. Ligand-induced conformational stabilization of proteins is a well-understood phenomenon in which substrates, inhibitors, cofactors, and even other proteins provide enhanced stability to proteins on binding. This phenomenon is based on the energetic coupling of the ligand-binding and protein-melting reactions. In an attempt to harness these biophysical properties for drug discovery, fully automated instrumentation was designed and implemented to perform miniaturized fluorescence-based thermal shift assays in a microplate format for the high throughput screening of compound libraries. Validation of this process and instrumentation was achieved by investigating ligand binding to more than 100 protein targets. The general applicability of the thermal shift screening strategy was found to be an important advantage because it circumvents the need to design and retool new assays with each new therapeutic target. Moreover, the miniaturized thermal shift assay methodology does not require any prior knowledge of a therapeutic target's function, making it ideally suited for the quantitative high throughput drug screening and evaluation of targets derived from genomics.

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Genome-Edited Coincidence and PMP22-HiBiT Fusion Reporter Cell Lines Enable an Artifact-Suppressive Quantitative High-Throughput Screening Strategy for PMP22 Gene-Dosage Disorder Drug Discovery

ACS Pharmacology & Translational Science ◽

10.1021/acsptsci.1c00110 ◽

2021 ◽

Author(s):

Natalia J. Martinez ◽

John C. Braisted ◽

Patricia K. Dranchak ◽

John J. Moran ◽

Hunter Larson ◽

...

Keyword(s):

Drug Discovery ◽

Cell Lines ◽

High Throughput ◽

High Throughput Screening ◽

Gene Dosage ◽

Screening Strategy ◽

Pmp22 Gene ◽

Reporter Cell

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A Fully Automated High-Throughput Flow Cytometry Screening System Enabling Phenotypic Drug Discovery

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555218773086 ◽

2018 ◽

Vol 23 (7) ◽

pp. 697-707 ◽

Cited By ~ 2

Author(s):

John Joslin ◽

James Gilligan ◽

Paul Anderson ◽

Catherine Garcia ◽

Orzala Sharif ◽

...

Keyword(s):

Flow Cytometry ◽

Drug Discovery ◽

High Throughput ◽

High Throughput Screening ◽

Screening System ◽

Preclinical Development ◽

Drug Identification ◽

Therapeutic Setting ◽

Compound Libraries ◽

Automated Platform

The goal of high-throughput screening is to enable screening of compound libraries in an automated manner to identify quality starting points for optimization. This often involves screening a large diversity of compounds in an assay that preserves a connection to the disease pathology. Phenotypic screening is a powerful tool for drug identification, in that assays can be run without prior understanding of the target and with primary cells that closely mimic the therapeutic setting. Advanced automation and high-content imaging have enabled many complex assays, but these are still relatively slow and low throughput. To address this limitation, we have developed an automated workflow that is dedicated to processing complex phenotypic assays for flow cytometry. The system can achieve a throughput of 50,000 wells per day, resulting in a fully automated platform that enables robust phenotypic drug discovery. Over the past 5 years, this screening system has been used for a variety of drug discovery programs, across many disease areas, with many molecules advancing quickly into preclinical development and into the clinic. This report will highlight a diversity of approaches that automated flow cytometry has enabled for phenotypic drug discovery.

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Drug discovery from Nature: automated high-quality sample preparation

Journal of Automated Methods and Management in Chemistry ◽

10.1155/s1463924600000249 ◽

2000 ◽

Vol 22 (5) ◽

pp. 149-157 ◽

Cited By ~ 12

Author(s):

Ralf Thiericke

Keyword(s):

Drug Discovery ◽

Sample Preparation ◽

High Throughput ◽

High Throughput Screening ◽

Solid Phase ◽

Structural Diversity ◽

Synthetic Compound ◽

Natural Origin ◽

Screening Programs ◽

Compound Libraries

Secondary metabolites from plants, animals and microorganisms have been proven to be an outstanding source for new and innovative drugs and show a striking structural diversity that supplements chemically synthesized compounds or libraries in drug discovery programs. Unfortunately, extracts from natural sources are usually complex mixtures of compounds:: often generated in time consuming and for the most part manual processes. As quality and quantity of the provided samples play a pivotal role in the success of high-throughput screening programs this poses serious problems. In order to make samples of natural origin competitive with synthetic compound libraries, we devised a novel, automated sample preparation procedure based on solid-phase extraction (SPE). By making use of a modified Zymark RapidTrace®SPE workstation an easy-to-handle and effective fractionation method has been developed which allows the generation of highquality samples from natural origin, fulfilling the requirements of an integration into high-throughput screening programs.

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Compartmentalization of enhanced biomolecular interactions for high-throughput drug screening in test tubes

10.1101/2020.01.19.911149 ◽

2020 ◽

Author(s):

Min Zhou ◽

Weiping Li ◽

Jian Li ◽

Leiming Xie ◽

Rongbo Wu ◽

...

Keyword(s):

Drug Discovery ◽

High Throughput ◽

Biomedical Research ◽

High Throughput Screening ◽

Biomolecular Interactions ◽

Compound Library ◽

Cellular Processes ◽

High Throughput Drug Screening ◽

Binding Partners ◽

Discovery Method

AbstractModification-dependent and -independent biomolecular interactions (BIs), including protein-protein, protein-DNA/RNA and protein-lipid, play crucial roles in all cellular processes. Dysregulation of BIs or malfunction of the associated enzymes results in various diseases, thus they are attractive targets for therapies. High-throughput screening (HTS) can greatly facilitate the discovery of drugs for these targets. Here we describe a HTS drug discovery method, called compartmentalization of enhanced biomolecular interactions in test tubes (CEBIT). CEBIT uses selective recruitment of biomolecules into phase separated compartments harboring their cognate binding partners as readouts. CEBIT were tailored to detect various BIs and associated modifying enzymes. Using CEBIT-based HTS assays, we successfully identified known inhibitors of the p53/MDM2 interaction and of SUV39H1 from a compound library. CEBIT is simple and versatile, and is likely to become a powerful tool for drug discovery and basic biomedical research.

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Natural Products in High Throughput Screening: Automated High-Quality Sample Preparation

CrossRef Listing of Deleted DOIs ◽

10.1177/108705719900400104 ◽

1999 ◽

Vol 4 (1) ◽

pp. 15-25 ◽

Cited By ~ 35

Author(s):

Ingrid Schmid ◽

Isabel Sattler ◽

Susanne Grabley ◽

Ralf Thiericke

Keyword(s):

Drug Discovery ◽

Sample Preparation ◽

High Throughput ◽

High Throughput Screening ◽

Solid Phase ◽

Structural Diversity ◽

High Quality ◽

Synthetic Compound ◽

Natural Origin ◽

Compound Libraries

At present, compound libraries from combinatorial chemistry are the major source for high throughput screening (HTS) programs in drug discovery. On the other hand, nature has been proven to be an outstanding source for new and innovative drugs. Secondary metabolites from plants, animals, and microorganisms show a striking structural diversity that supplements chemically synthesized compounds or libraries in drug discovery programs. Unfortunately, extracts from natural sources are usually complex mixtures of compounds, often generated in time-consuming and, for the most part, manual processes. Because quality and quantity of the provided samples play a pivotal role in the success of HTS programs, this poses serious problems. In order to make samples of natural origin competitive with synthetic compound libraries, we devised a novel, automated sample preparation procedure based on solid-phase extraction (SPE). By making use of modified Zymark (Hopkinton, MA) RapidTrace® SPE workstations, we developed an easy-to-handle and effective fractionation method that generates high-quality samples from natural origin, fulfilling the requirements for an integration in high throughput drug discovery programs.

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Statistical models for identifying frequent hitters in high throughput screening

Scientific Reports ◽

10.1038/s41598-020-74139-0 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Samuel Goodwin ◽

Golnaz Shahtahmassebi ◽

Quentin S. Hanley

Keyword(s):

Drug Discovery ◽

High Throughput ◽

High Throughput Screening ◽

Negative Binomial ◽

Data Sets ◽

Individual Compound ◽

Data Set ◽

Binomial Distributions ◽

Compound Libraries ◽

Large Numbers

Abstract High throughput screening (HTS) interrogates compound libraries to find those that are “active” in an assay. To better understand compound behavior in HTS, we assessed an existing binomial survivor function (BSF) model of “frequent hitters” using 872 publicly available HTS data sets. We found large numbers of “infrequent hitters” using this model leading us to reject the BSF for identifying “frequent hitters.” As alternatives, we investigated generalized logistic, gamma, and negative binomial distributions as models for compound behavior. The gamma model reduced the proportion of both frequent and infrequent hitters relative to the BSF. Within this data set, conclusions about individual compound behavior were limited by the number of times individual compounds were tested (1–1613 times) and disproportionate testing of some compounds. Specifically, most tests (78%) were on a 309,847-compound subset (17.6% of compounds) each tested ≥ 300 times. We concluded that the disproportionate retesting of some compounds represents compound repurposing at scale rather than drug discovery. The approach to drug discovery represented by these 872 data sets characterizes the assays well by challenging them with many compounds while each compound is characterized poorly with a single assay. Aggregating the testing information from each compound across the multiple screens yielded a continuum with no clear boundary between normal and frequent hitting compounds.

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The MALDI TOF E2/E3 ligase assay as an universal tool for drug discovery in the ubiquitin pathway

10.1101/224600 ◽

2017 ◽

Author(s):

Virginia De Cesare ◽

Clare Johnson ◽

Victoria Barlow ◽

James Hastie ◽

Axel Knebel ◽

...

Keyword(s):

Drug Discovery ◽

High Throughput ◽

High Throughput Screening ◽

E3 Ligase ◽

Ubiquitin Pathway ◽

E3 Ligases ◽

Maldi Tof ◽

Compound Libraries ◽

Ubiquitin System ◽

Conjugating Enzymes

AbstractIn many diseases, components of the ubiquitin system - such as E2/E3 ligases and deubiquitylases - are dysregulated. The ubiquitin system has therefore become an emergent target for the treatment of a number of diseases, including cancer, neurodegeneration and autoimmunity. Despite of the efforts in this field, primary screenings of compound libraries to individuate new potential therapeutic molecules targeting the ubiquitin pathway have been strongly limited by the lack of robust and fast high-throughput assays. Here we report the first label-free high-throughput screening (HTS) assay for ubiquitin E2 conjugating enzymes and E3 ligases based on Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight (MALDI TOF) mass spectrometry. The MALDI TOF E2/E3 assay allows us to test E2 conjugating enzymes and E3 ligases for their ubiquitin transfer activity, to identify E2/E3 active pairs, inhibitor potency and specificity and to screen compound libraries in vitro without synthesis of chemical or fluorescent probes. We demonstrate that the MALDI TOF E2/E3 assay is a universal tool for drug discovery screening in the ubiquitin pathway as it is suitable for working with all E3 ligase families and requires a reduced amount of reagents, compared to standard biochemical assays.

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