High-Throughput Implementation of the NanoBRET Target Engagement Intracellular Kinase Assay to Reveal Differential Compound Engagement by SIK2/3 Isoforms

The real-time quantification of target engagement (TE) by small-molecule ligands in living cells remains technically challenging. Systematic quantification of such interactions in a high-throughput setting holds promise for identification of target-specific, potent small molecules within a pathophysiological and biologically relevant cellular context. The salt-inducible kinases (SIKs) belong to a subfamily of the AMP-activated protein kinase (AMPK) family and are composed of three isoforms in humans (SIK1, SIK2, and SIK3). They modulate the production of pro- and anti-inflammatory cytokines in immune cells. Although pan-SIK inhibitors are sufficient to reverse SIK-dependent inflammatory responses, the apparent toxicity associated with SIK3 inhibition suggests that isoform-specific inhibition is required to realize therapeutic benefit with acceptable safety margins. Here, we used the NanoBRET TE intracellular kinase assay, a sensitive energy transfer technique, to directly measure molecular proximity and quantify TE in HEK293T cells overexpressing SIK2 or SIK3. Our 384-well high-throughput screening of 530 compounds demonstrates that the NanoBRET TE intracellular kinase assay was sensitive and robust enough to reveal differential engagement of candidate compounds with the two SIK isoforms and further highlights the feasibility of high-throughput implementation of NanoBRET TE intracellular kinase assays for target-driven small-molecule screening.

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High-Throughput Screening of Small Molecule Ligands Targeted to Live Bacteria Surface

Analytical Chemistry ◽

10.1021/ac303199x ◽

2013 ◽

Vol 85 (7) ◽

pp. 3508-3514 ◽

Cited By ~ 9

Author(s):

Jeong Heon Lee ◽

Sunny Park ◽

Hoon Hyun ◽

Mark W. Bordo ◽

Rafiou Oketokoun ◽

...

Keyword(s):

Small Molecule ◽

High Throughput ◽

High Throughput Screening ◽

Small Molecule Ligands ◽

Live Bacteria

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SCAN ™—A High-Throughput Assay for Detecting Small Molecule Binding to RNA Targets

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057108330111 ◽

2009 ◽

Vol 14 (3) ◽

pp. 219-229 ◽

Cited By ~ 6

Author(s):

Chris Baugh ◽

Shaohui Wang ◽

Bin Li ◽

James R. Appleman ◽

Peggy A. Thompson

Keyword(s):

Small Molecule ◽

High Throughput ◽

High Throughput Screening ◽

Screening Assay ◽

Laboratory Equipment ◽

Rna Targets ◽

High Throughput Screening Assay ◽

Small Molecule Ligands ◽

Hcv Ires ◽

High Throughput Assay

A novel optical-based high-throughput screening technology has been developed for increasing the rate of discovering chemical leads against RNA targets. SCAN™ ( Screen for Compounds with Affinity for Nucleic Acids) is an affinity-based assay that identifies small molecules that bind and recognize structured RNA elements. This technology provides the opportunity to conduct high-throughput screening of a new class of targets—RNA. SCAN™ offers many attractive features including a simple homogeneous format, low screening costs, and the ability to use common laboratory equipment. A SCAN™ assay was developed for the HCV IRES Loop IIId RNA domain. A high-throughput screen of our entire compound library resulted in the identification of small molecule ligands that bind to Loop IIId. The Z′ values were greater than 0.8, showing this to be a robust high-throughput screening assay. A correlation between SCAN™ EC50 and KD values is reported suggesting the ability to use the assay for compound optimization. ( Journal of Biomolecular Screening 2009:219-229)

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Development and Implementation of an HTS-Compatible Assay for the Discovery of Selective Small-Molecule Ligands for Pre-microRNAs

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555217717944 ◽

2017 ◽

Vol 23 (1) ◽

pp. 47-54 ◽

Cited By ~ 4

Author(s):

Daniel A. Lorenz ◽

Steve Vander Roest ◽

Martha J. Larsen ◽

Amanda L. Garner

Keyword(s):

Small Molecules ◽

Natural Product ◽

Small Molecule ◽

High Throughput ◽

High Throughput Screening ◽

Two Dimensional ◽

Small Molecule Ligands ◽

Gene Regulatory ◽

Regulatory Rnas ◽

Target Rna

microRNAs (miRNAs) are small gene regulatory RNAs, and their expression has been found to be dysregulated in a number of human diseases. To facilitate the discovery of small molecules capable of selectively modulating the activity of a specific miRNA, we have utilized new high-throughput screening technology targeting Dicer-mediated pre-miRNA maturation. Pilot screening of ~50,000 small molecules and ~33,000 natural product extract libraries against pre-miR-21 processing indicated the potential of our assay for this goal, yielding a campaign Z′ factor of 0.52 and an average plate signal-to-background (S/B) ratio of 13. Using two-dimensional screening against a second pre-miRNA, pre-let-7d, we evaluated the selectivity of confirmed hits. The results presented demonstrate how high-throughput screening can be used to identify selective small molecules for a target RNA.

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Discovery and Characterization of Orthosteric and Allosteric Muscarinic M2 Acetylcholine Receptor Ligands by Affinity Selection-Mass Spectrometry

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057105284340 ◽

2005 ◽

Vol 11 (2) ◽

pp. 194-207 ◽

Cited By ~ 32

Author(s):

Charles E. Whitehurst ◽

Naim Nazef ◽

D. Allen Annis ◽

Yongmin Hou ◽

Denise M. Murphy ◽

...

Keyword(s):

Mass Spectrometry ◽

Acetylcholine Receptor ◽

Small Molecule ◽

High Throughput ◽

High Throughput Screening ◽

Size Exclusion ◽

Affinity Selection ◽

Binding Properties ◽

Small Molecule Ligands

Screening assays using target-based affinity selection coupled with high-sensitivity detection technologies to identify small-molecule hits from chemical libraries can provide a useful discovery approach that complements traditional assay systems. Affinity selection-mass spectrometry (AS-MS) is one such methodology that holds promise for providing selective and sensitive high-throughput screening platforms. Although AS-MS screening platforms have been used to discover small-molecule ligands of proteins from many target families, they have not yet been used routinely to screen integral membrane proteins. The authors present a proof-of-concept study using size exclusion chromatography coupled to AS-MS to perform a primary screen for small-molecule ligands of the purified muscarinic M2 acetylcholine receptor, a G-protein-coupled receptor. AS-MS is used to characterize the binding mechanisms of 2 newly discovered ligands. NGD-3350 is a novel M2-specific orthosteric antagonist of M2 function. NGD-3366 is an allosteric ligand with binding properties similar to the allosteric antagonist W-84, which decreases the dissociation rate of N-methyl-scopolamine from the M2 receptor. Binding properties of the ligands discerned from AS-MS assays agree with those from in vitro biochemical assays. The authors conclude that when used with appropriate small-molecule libraries, AS-MS may provide a useful high-throughput assay system for the discovery and characterization of all classes of integral membrane protein ligands, including allosteric modulators.

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Identifying Druglike Inhibitors of Myelin-Reactive T Cells by Phenotypic High-Throughput Screening of a Small-Molecule Library

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057107301272 ◽

2007 ◽

Vol 12 (4) ◽

pp. 481-489 ◽

Cited By ~ 5

Author(s):

Christina Rossi ◽

Deepa Padmanaban ◽

Jake Ni ◽

Li-An Yeh ◽

Marcie A. Glicksman ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Transgenic Mice ◽

Small Molecule ◽

High Throughput ◽

Proinflammatory Cytokines ◽

High Throughput Screening ◽

Inflammatory Responses ◽

Critical Role ◽

High Throughput Screening Assay

Inflammatory T cells that are reactive to myelin protein components of the CNS play a critical role in the pathogenesis of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). The authors have previously generated mice that predominantly harbor T cells transgenic for a T-cell receptor (TCR) that is specific to the myelin proteolipid protein (PLP) 139-151 and that spontaneously develop MS-like paralysis. T cells from healthy transgenic mice respond to stimulation with PLP139-151 in a highly specific manner by proliferation and secretion of proinflammatory cytokines such as interleukin (IL)—2 and interferon (INF)—γ in vitro. To identify druglike compounds that may inhibit inflammatory T-cell responses, the authors have developed a high-throughput screening assay with primary T cells from PLP TCR transgenic mice. They have screened 41,184 small-molecule compounds that follow Lipinski's rules for their inhibitory activity on the proliferation and secretion of proinflammatory cytokines in PLP-reactive T cells. To this end, the screen identified 6 nontoxic compounds with a molecular weight <500 that inhibited inflammatory responses in PLP-reactive T cells in a concentration-dependent fashion. The identified compounds represent valid leads that may be developed into novel therapeutics for MS that could be administered orally. ( Journal of Biomolecular Screening 2007:481-489)

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Faculty Opinions recommendation of High-throughput screening yields several small-molecule inhibitors of repeat-associated non-AUG translation.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.736805126.793566632 ◽

2019 ◽

Author(s):

John Lowe

Keyword(s):

Small Molecule ◽

High Throughput ◽

High Throughput Screening ◽

Small Molecule Inhibitors

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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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A small molecule UPR modulator for diabetes identified by high throughput screening

Acta Pharmaceutica Sinica B ◽

10.1016/j.apsb.2021.05.018 ◽

2021 ◽

Author(s):

Valeria Marrocco ◽

Tuan Tran ◽

Siying Zhu ◽

Seung Hyuk Choi ◽

Ana M. Gamo ◽

...

Keyword(s):

Small Molecule ◽

High Throughput ◽

High Throughput Screening

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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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