Establishment and Application of a High Throughput Model for Rho Kinase Inhibitors Screening Based on Fluorescence Polarization

Duan, Weigang; Sun, Lixin; Li, Jun; Wu, Xudong; Zhang, Luyong; Yan, Ming

doi:10.1248/bpb.29.1138

Cited by 5 publications

(3 citation statements)

References 23 publications

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“…32) In the case of false negatives and referring to the report by Duan et al 33) , we suggest the IC 50 threshold for candidate compounds should be less than 10 mM. To detect hits directly, the standard curve need not be established each time, and the hits can be detected from the original data (EIA absorbance) by observing whether the absorbance caused by a compound (10 mM) is less than that by the positive control (NS398 2 mM).…”

Section: Discussionmentioning

confidence: 99%

Screening Method for Nonsteroidal Antiinflammatory Drugs Based on the Cyclooxygenase 2 Pathway Activated by Serum-Free Stimulation in A549 Cells

Yao

Duan

et al. 2007

YAKUGAKU ZASSHI

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Cyclooxygenase 2 (COX-2) pathway inhibitors were regarded as promising nonsteroidal antiin‰ammatory drugs (NSAIDs). We discovered that the COX-2 pathway in A549 cells, a human lung cancer cell line, was activated by serumfree stimulation, and a drug screening model for NSAIDs was established based on this principle with simple performance and su‹cient reliability. The COX-2 pathway was activated by treating with serum-free medium for 12 h. The activated cells were incubated with NS398 (selective COX-2 inhibitor), SC560 (selective COX-1 inhibitor), acetyl salicylic acid (ASA) (nonselective COX inhibitor) at 37°C for 15 min. Then the cells were incubated with 10 mM of arachidonic acid (AA) for another 30 min prostaglandin E 2 and 6-keto-prostaglandin F 1a were assayed in an enzyme immunoassay (EIA). The results showed that the COX-2 pathway was dominant in A549 cells whether activated by serum-free medium or not, and the COX-1 pathway could be ignored. The model accepted the positive inhibition threshold as NS398 2 mM; if a compound (10 mM) inhibited COX-2 pathway more than NS398 (2 mM), it was regarded as a hit. The COX-2 pathway inhibition experiment showed that the Z`-factor of the screening model was 0.62, which suggests that the model is suitable for COX-2 pathway inhibitor screening.

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Section: Discussionmentioning

confidence: 99%

Screening Method for Nonsteroidal Antiinflammatory Drugs Based on the Cyclooxygenase 2 Pathway Activated by Serum-Free Stimulation in A549 Cells

Yao

Duan

et al. 2007

YAKUGAKU ZASSHI

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“…Fluorescence polarization assays have been developed to detect various biological events such as phosphorylation, proteolytic cleavage, single nucleotide polymorphism detection, cAMP production, protein-protein interactions, and protein-DNA interactions [28,29,31,32,[37][38][39][40][41]. This article focuses on our development and validation of a ligand displacement assay to screen for inhibitors of RGS12/G i1 and GPSM2/G i1 interactions (Fig.…”

Section: Introductionmentioning

confidence: 99%

A High Throughput Fluorescence Polarization Assay for Inhibitors of the GoLoco Motif/G-alpha Interaction

Kimple¹,

Yasgar²,

Hughes³

et al. 2008

CCHTS

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The GoLoco motif is a short Galpha-binding polypeptide sequence. It is often found in proteins that regulate cell-surface receptor signaling, such as RGS12, as well as in proteins that regulate mitotic spindle orientation and force generation during cell division, such as GPSM2/LGN. Here, we describe a high throughput fluorescence polarization (FP) assay using fluorophore-labeled GoLoco motif peptides for identifying inhibitors of the GoLoco motif interaction with the G-protein alpha subunit Galpha (i1). The assay exhibits considerable stability over time and is tolerant to DMSO up to 5%. The Z'-factors for robustness of the GPSM2 and RGS12 GoLoco motif assays in a 96-well plate format were determined to be 0.81 and 0.84, respectively; the latter assay was run in a 384-well plate format and produced a Z'-factor of 0.80. To determine the screening factor window (Z-factor) of the RGS12 GoLoco motif screen using a small molecule library, the NCI Diversity Set was screened. The Z-factor was determined to be 0.66, suggesting that this FP assay would perform well when developed for 1,536-well format and scaled up to larger libraries. We then miniaturized to a 4 microL final volume a pair of FP assays utilizing fluorescein- (green) and rhodamine- (red) labeled RGS12 GoLoco motif peptides. In a fully-automated run, the Sigma-Aldrich LOPAC(1280) collection was screened three times with every library compound being tested over a range of concentrations following the quantitative high throughput screening (qHTS) paradigm; excellent assay performance was noted with average Z-factors of 0.84 and 0.66 for the green- and red-label assays, respectively.

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“…Cell Culture. PC12 cells were incubated in Ham's F12K medium with 2 mM l ‐glutamine adjusted to contain 1.5 g/l sodium bicarbonate with 15% horse serum and 2.5% foetal bovine serum in an atmosphere containing 5% CO 2 and 95% air at 37°[17]. When PC12 cells were about to occupy 80% of the flask, they were dispersed and seeded in 96‐well plates coated with 0.01% polylysine.…”

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confidence: 99%

Rho Kinase Inhibitor Y‐27632 Down‐Regulates Norepinephrine Synthesis and Release in PC12 Cells

Duan

Shang

Jiang

et al. 2009

Basic Clin Pharma Tox

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Rho kinase inhibition is beneficial for neurite outgrowth and nerve disorders, and the Rho kinase inhibitors have been regarded as promising agents to treat neural diseases. The main aim of the study was to elucidate how Rho kinase inhibitor Y-27632 regulates neurotransmitter norepinephrine synthesis and release in PC12 cells when neurite outgrowth was induced. PC12 cells were treated with Y-27632 for 6 days. The amount of norepinephrine synthesized in PC12 cells and the amount released evoked by acetylcholine or by KCl were determined by norepinephrine enzyme-linked immunosorbent assay kits. The results showed that the amount of norepinephrine both synthesized and released was down-regulated with a concentration-dependent relationship. Further results of Western blotting found that the protein expression of tyrosine hydroxylase and synapsin I (especially its active form, synapsin I phosphoSer603) was also down-regulated, which were directly related to synthesis and release of norepinephrine, respectively. All the results suggest that Y-27632 is able to downregulate norepinephrine synthesis and release, the direct mechanism of which may be associated with down-regulation on expression of some proteins, including tyrosine hydroxylase and synapsin I.

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Establishment and Application of a High Throughput Model for Rho Kinase Inhibitors Screening Based on Fluorescence Polarization

Cited by 5 publications

References 23 publications

Screening Method for Nonsteroidal Antiinflammatory Drugs Based on the Cyclooxygenase 2 Pathway Activated by Serum-Free Stimulation in A549 Cells

Screening Method for Nonsteroidal Antiinflammatory Drugs Based on the Cyclooxygenase 2 Pathway Activated by Serum-Free Stimulation in A549 Cells

A High Throughput Fluorescence Polarization Assay for Inhibitors of the GoLoco Motif/G-alpha Interaction

Rho Kinase Inhibitor Y‐27632 Down‐Regulates Norepinephrine Synthesis and Release in PC12 Cells

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