scholarly journals Development of an Assay to Screen for Inhibitors of Tau Phosphorylation by Cdk5

2004 ◽  
Vol 9 (2) ◽  
pp. 122-131 ◽  
Author(s):  
Jae Suk Ahn ◽  
Andrea Musacchio ◽  
Marina Mapelli ◽  
Jake Ni ◽  
Leonard Scinto ◽  
...  
Keyword(s):  
Kinetic Parameters ◽  
Adenosine Triphosphate ◽  
Immunosorbent Assay ◽  
High Throughput ◽  
Rate Constants ◽  
Colorimetric Assay ◽  
Tau Phosphorylation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Site Specific ◽  
High Throughput Assay

A high-throughput assay for tau phosphorylation by cdk5/p25 is described. Full-length recombinant tau was used as a substrate in the presence of saturating adenosine triphosphate (ATP). Using PHF-1, an antibody directed specifically against 2 tau phosphorylation epitopes (serine 396 and serine 404), an enzyme-linked immunosorbent assay (ELISA)- based colorimetric assay was formatted in 384-well plates. The assay was validated by measuring kinetic parameters for cdk5/p25 catalysis and known inhibitors. Rate constants for the site-specific phosphorylations at the PHF-1 epitopes were determined and suggested preferential phosphorylation at these sites. The performance of this assay in a high-throughput format was demonstrated and used to identify inhibitors of tau phosphorylation at specific epitopes phosphorylated by cdk5/p25. ( Journal of Biomolecular Screening 2004:122-131)

An Enzyme-Linked Immunosorbent Assay for Protein Kinase D Activity Using Phosphorylation Site-Specific Antibodies

2007 ◽  
Vol 5 (5) ◽  
pp. 637-644 ◽  
Author(s):  
An Rykx ◽  
Sadia Vancauwenbergh ◽  
Line De Kimpe ◽  
Katrien Janssens ◽  
Sandy Vandoninck ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Immunosorbent Assay ◽  
Phosphorylation Site ◽  
Enzyme Linked Immunosorbent Assay ◽  
Protein Kinase D ◽  
Site Specific ◽  
Specific Antibodies

scholarly journals Identification and Characterization of a New Series of Ghrelin O-Acyl Transferase Inhibitors

2017 ◽  
Vol 23 (2) ◽  
pp. 154-163 ◽  
Author(s):  
Mariko Yoneyama-Hirozane ◽  
Kohei Deguchi ◽  
Takeshi Hirakawa ◽  
Tsuyoshi Ishii ◽  
Tomoyuki Odani ◽  
...  
Keyword(s):  
High Throughput ◽  
Inhibitory Activity ◽  
High Throughput Screening ◽  
Enzyme Linked Immunosorbent Assay ◽  
Acyl Transferase ◽  
Lead Compound ◽  
Time Resolved ◽  
Acyl Ghrelin ◽  
Antiobesity Drugs ◽  
High Throughput Assay

Ghrelin O-acyl transferase (GOAT; MBOAT4) catalyzes O-acylation at serine-3 of des-acyl ghrelin. Acyl ghrelin is secreted by stomach X/A-like cells and plays a role in appetite and metabolism. Therefore, GOAT has been expected to be a novel antiobesity target because it is responsible for acyl ghrelin production. Here, we report homogeneous time-resolved fluorescence (HTRF) and enzyme-linked immunosorbent assay (ELISA) methods utilizing human GOAT-expressing microsomes as a novel high-throughput assay system for the discovery of hit compounds and optimization of lead compounds. Hit compounds exemplified by compound A (2-[(2,4-dichlorobenzyl)sulfanyl]-1,3-benzoxazole-5-carboxylic acid) were identified by high-throughput screening using the HTRF assay and confirmed to have GOAT inhibitory activity using the ELISA. Based on the hit compound information, the novel lead compound (compound B, (4-chloro-6-{[2-methyl-6-(trifluoromethyl)pyridin-3-yl]methoxy}-1-benzothiophen-3-yl)acetic acid) was synthesized and exhibited potent GOAT inhibition with oral bioavailability. Both the hit compound and lead compound showed octanoyl-CoA competitive inhibitory activity. Moreover, these two compounds decreased acyl ghrelin production in the stomach of mice after their oral administration. These novel findings demonstrate that GOAT is a druggable target, and its inhibitors are promising antiobesity drugs.

A High Throughput Enzyme-Linked Immunosorbent Assay for Inhibitors of the Interaction Between Retinoblastoma Protein and the Leu-X-Cys-X-Glu Motif

1997 ◽  
Vol 2 (4) ◽  
pp. 207-211 ◽  
Author(s):  
Victoria Alice Ellsmore ◽  
Al Peng Teoh ◽  
Arasu Ganesan
Keyword(s):  
Immunosorbent Assay ◽  
High Throughput ◽  
High Throughput Screening ◽  
Microtiter Plate ◽  
Enzyme Linked Immunosorbent Assay ◽  
Retinoblastoma Tumor Suppressor ◽  
Multiple Antigenic Peptide ◽  
Peptide Map ◽  
Retinoblastoma Tumor ◽  
Lxcxe Motif

A 96-well enzyme-linked immunosorbent assay was developed to discover compounds that inhibit the binding of the Leu-X-Cys-X-Glu (LXCXE) motif to the retinoblastoma tumor suppressor protein (pRB). The assay uses a LXCXE-containing multiple antigenic peptide (MAP) which is immobilized on a microtiter plate. A truncated form of pRB is added and the amount bound detected by a monoclonal antibody. This rapid assay was employed in high throughput screening of crude natural product extracts and discrete compounds.

scholarly journals Rapid and Automated Fluorescence-Linked Immunosorbent Assay for High-Throughput Screening of Hiv-1 Fusion Inhibitors Targeting gp41

2003 ◽  
Vol 8 (6) ◽  
pp. 685-693 ◽  
Author(s):  
Shuwen Liu ◽  
Louise Boyer-Chatenet ◽  
Hong Lu ◽  
Shibo Jiang
Keyword(s):  
Immunosorbent Assay ◽  
High Throughput ◽  
High Throughput Screening ◽  
Sandwich Elisa ◽  
Enzymatic Reaction ◽  
Heptad Repeat ◽  
Enzyme Linked Immunosorbent Assay ◽  
Helix Bundle ◽  
Fusion Inhibitors ◽  
Hiv 1

The human immuno deficiency virus type 1 (HIV-1) envelope glycoprotein gp41 plays animportant role in the virus entry. During the process of fusion between the viral and target cell membranes, the N-and C-terminal heptad repeat (HR) regions of the gp41 extracellular domain associate to form a 6-helical bundle, corresponding to the fusion-active gp41 core. Any compound that blocks the gp41 6-helix bundle formation between the N- and C-peptides, which are derived from the N- and C-terminal HR regions, respectively, may inhibit HIV-1 mediated membrane fusion. Based on this principle, we previously established a sandwich enzyme-linked immunosorbent assay (ELISA) for drug screening by using the N-peptide N36 and the C-peptide C34 and a monoclonal antibody (NC-1) which specifically recognizes the gp41 6-helix bundle. In the present study, a fluorescence-linked immunosorbent assay (FLISA) was developed by using fluorescein isothiocyanate (FITC)-conjugated C34 to replace C34 and by directly detecting fluorescence intensity instead of more complicated enzymatic reaction. Compared with the sandwich ELISA, this FLISA has similar sensitivity and specificity, but it is much more rapid, economic and convenient. Using an Integrated Robotic Sample Processing System, this assay has been applied for high-throughput screening of organic compounds on a large scale for HIV-1 fusion inhibitors targeting gp41.

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