Identification of a calcium channel modulator using a high throughput yeast two-hybrid screen

Young, Kimberly A.; Lin, Stephen; Sun, Lucy; Lee, Eunice; Modi, Mita; Hellings, Samuel; Husbands, Morris; Ozenberger, B A; Franco, Rodrigo

doi:10.1038/nbt1098-946

Cited by 112 publications

(70 citation statements)

References 37 publications

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“…[1][2][3][4][5] When the three-dimensional structure of the target is known or can be modeled, virtual screening using molecular docking can also be used to discover new lead compounds. [6][7][8][9][10] In principle, HTS should discover all of the interesting ligands in a database, whereas the predictions of computer-based docking screens are less reliable and must be tested.…”

Section: Introductionmentioning

confidence: 99%

Molecular Docking and High-Throughput Screening for Novel Inhibitors of Protein Tyrosine Phosphatase-1B

et al. 2002

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High-throughput screening (HTS) of compound libraries is used to discover novel leads for drug development. When a structure is available for the target, computer-based screening using molecular docking may also be considered. The two techniques have rarely been used together on the same target. The opportunity to do so presented itself in a project to discover novel inhibitors for the enzyme protein tyrosine phosphatase-1B (PTP1B), a tyrosine phosphatase that has been implicated as a key target for type II diabetes. A corporate library of approximately 400 000 compounds was screened using high-throughput experimental techniques for compounds that inhibited PTP1B. Concurrently, molecular docking was used to screen approximately 235 000 commercially available compounds against the X-ray crystallographic structure of PTP1B, and 365 high-scoring molecules were tested as inhibitors of the enzyme.Of approximately 400 000 molecules tested in the high-throughput experimental assay, 85 (0.021%) inhibited the enzyme with IC 50 values less than 100 µM; the most active had an IC 50 value of 4.2 µM. Of the 365 molecules suggested by molecular docking, 127 (34.8%) inhibited PTP1B with IC 50 values less than 100 µM; the most active of these had an IC 50 of 1.7 µM. Structure-based docking therefore enriched the hit rate by 1700-fold over random screening. The hits from both the high-throughput and docking screens were dissimilar from phosphotyrosine, the canonical substrate group for PTP1B; the two hit lists were also very different from each other. Surprisingly, the docking hits were judged to be more druglike than the HTS hits. The diversity of both hit lists and their dissimilarity from each other suggest that docking and HTS may be complementary techniques for lead discovery.

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Section: Introductionmentioning

confidence: 99%

Molecular Docking and High-Throughput Screening for Novel Inhibitors of Protein Tyrosine Phosphatase-1B

et al. 2002

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“…The interaction of human calcium channel subunits α1B and β3 has been coupled to a marker conferring cycloheximide sensitivity (Young et al 1998). A screen of >150 000 compounds identified 10 compounds that rescued growth of the α1B and β3 strain, but not a control strain in the presence of cycloheximide.…”

Section: Y E a S T A S A T R A N S A C T I Va T I O N P L A T F O R Mmentioning

confidence: 99%

The utility of yeast as a tool for cell-based, target-directed high-throughput screening

Norcliffe¹,

Álvarez-Ruíz

Martín-Plaza

et al. 2013

Parasitology

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“…17 To realize the speed and automa-tion necessary to aggressively pursue potential modulators of protein-protein interactions, YTH-based assays would benefit from a quantitative rapid reporter system that could be adapted into a liquid-based HTS assay format. Because the majority of the YTH reporters currently in use have an assay window of several days, 23 we developed and validated the functionality of several luciferase reporter genes in various YTH interactions.…”

Section: Design Of Dual Luciferase Yth Assaymentioning

confidence: 99%

“…12 Previous YTH screening methods used an agar-based platform. 17 To develop a YTH-based assay format amenable for high throughput, we investigated luci-ferase reporter systems. Despite being commonly used as a reporter gene in mammalian cell-based systems, 18 luciferase (luc) has not been used in an analogous manner in yeast.…”

mentioning

confidence: 99%

A Dual Luciferase Multiplexed High-Throughput Screening Platform for Protein-Protein Interactions

Nieuwenhuijsen

Huang²,

Wang

et al. 2003

SLAS Discovery

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To study the biology of regulators of G-protein signaling (RGS) proteins and to facilitate the identification of small molecule modulators of RGS proteins, the authors recently developed an advanced yeast 2-hybrid (YTH) assay format for GαZ and RGS-Z1. Moreover, they describe the development of a multiplexed luciferase-based assay that has been successfully adapted to screen large numbers of small molecule modulators of protein-protein interactions. They generated and evaluated 2 different luciferase reporter gene systems for YTH interactions, a Gal4 responsive firefly luciferase reporter gene and a Gal4 responsive Renilla luciferase reporter gene. Both the firefly and Renilla luciferase reporter genes demonstrated a 40-to 50-fold increase in luminescence in strains expressing interacting YTH fusion proteins versus negative control strains. Because the firefly and Renilla luciferase proteins have different substrate specificity, the assays were multiplexed. The multiplexed luciferase-based YTH platform adds speed, sensitivity, simplicity, quantification, and efficiency to YTH high-throughput applications and therefore greatly facilitates the identification of small molecule modulators of protein-protein interactions as tools or potential leads for drug discovery efforts. (Journal of Biomolecular Screening 2003:676-684)

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Identification of a calcium channel modulator using a high throughput yeast two-hybrid screen

Cited by 112 publications

References 37 publications

Molecular Docking and High-Throughput Screening for Novel Inhibitors of Protein Tyrosine Phosphatase-1B

Molecular Docking and High-Throughput Screening for Novel Inhibitors of Protein Tyrosine Phosphatase-1B

The utility of yeast as a tool for cell-based, target-directed high-throughput screening

A Dual Luciferase Multiplexed High-Throughput Screening Platform for Protein-Protein Interactions

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