Human Adenine Nucleotide Translocase (ANT) Modulators Identified by High-Throughput Screening of Transgenic Yeast

Transport of ADP and ATP across mitochondria is one of the primary points of regulation to maintain cellular energy homeostasis. This process is mainly mediated by adenine nucleotide translocase (ANT) located on the mitochondrial inner membrane. There are four human ANT isoforms, each having a unique tissue-specific expression pattern and biological function, highlighting their potential as drug targets for diverse clinical indications, including male contraception and cancer. In this study, we present a novel yeast-based high-throughput screening (HTS) strategy to identify compounds inhibiting the function of ANT. Yeast strains generated by deletion of endogenous proteins with ANT activity followed by insertion of individual human ANT isoforms are sensitive to cell-permeable ANT inhibitors, which reduce proliferation. Screening hits identified in the yeast proliferation assay were characterized in ADP/ATP exchange assays employing recombinant ANT isoforms expressed in isolated yeast mitochondria and Lactococcus lactis as well as by oxygen consumption rate in mammalian cells. Using this approach, closantel and CD437 were identified as broad-spectrum ANT inhibitors, whereas leelamine was found to be a modulator of ANT function. This yeast “knock-out/knock-in” screening strategy is applicable to a broad range of essential molecular targets that are required for yeast survival.

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Development of a Novel High-Throughput Screen and Identification of Small-Molecule Inhibitors of the Gα–RGS17 Protein–Protein Interaction Using AlphaScreen

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057111410427 ◽

2011 ◽

Vol 16 (8) ◽

pp. 869-877 ◽

Cited By ~ 13

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.

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

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/24725552211026245 ◽

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.

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

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555219873068 ◽

2019 ◽

Vol 25 (1) ◽

pp. 9-20 ◽

Cited By ~ 2

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.

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A high-throughput screening strategy identifies cardiotonic steroids as alternative splicing modulators

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0801661105 ◽

2008 ◽

Vol 105 (32) ◽

pp. 11218-11223 ◽

Cited By ~ 87

Author(s):

P. Stoilov ◽

C.-H. Lin ◽

R. Damoiseaux ◽

J. Nikolic ◽

D. L. Black

Keyword(s):

Alternative Splicing ◽

High Throughput ◽

High Throughput Screening ◽

Screening Strategy

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

Micromachines ◽

10.3390/mi12060639 ◽

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.

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