+ Site Statistics
+ Search Articles
+ PDF Full Text Service
How our service works
Request PDF Full Text
+ Follow Us
Follow on Facebook
Follow on Twitter
Follow on LinkedIn
+ Subscribe to Site Feeds
Most Shared
PDF Full Text
+ Translate
+ Recently Requested

High-throughput screening for the identification of small-molecule inhibitors of retinoblastoma protein phosphorylation in cells



High-throughput screening for the identification of small-molecule inhibitors of retinoblastoma protein phosphorylation in cells



Analytical Biochemistry 320(1): 66-74



The tumor suppressor protein, pRb, regulates progression through the G1 phase of the cell cycle by its ability to bind to and regulate the activity of a variety of transcription factors. This function of pRb is disabled through its phosphorylation by the cyclin-dependent kinase (CDK) family of serine/threonine kinases. In many human cancers, genetic alteration such as loss of CDK inhibitor function and deregulated G1 cyclin expression leads to inappropriate phosphorylation and hence inactivation of this tumor suppressor. Identification of cell-permeable small molecules that block pRb phosphorylation in these tumors could therefore lead to development of an effective anticancer treatment. As a result, we have developed a high-throughput assay to detect changes in the level of pRb phosphorylation in cells. Signal detection is by a time-resolved fluorescence-based cellular immunosorbant assay on a fixed monolayer of cells. This comprises a mouse monoclonal antibody that recognizes the phosphorylated form of serine 608 on pRb, a known site of CDK phosphorylation. and a Europium-labeled secondary antibody for signal detection. The assay is reproducible and amenable to automation and has been used to screen 2000 compounds in a search for cell-permeable small molecules that will block pRb phosphorylation.

Please choose payment method:






(PDF emailed within 0-6 h: $19.90)

Accession: 010753827

Download citation: RISBibTeXText

PMID: 12895470

DOI: 10.1016/s0003-2697(03)00349-x


Related references

High-throughput screening for the identification of small-molecule inhibitors of the flaviviral protease. Antiviral Research 134: 6-16, 2016

Identification of small molecule inhibitors of p27(Kip1) ubiquitination by high-throughput screening. Cancer Science 104(11): 1461-1467, 2014

A high throughput screening assay system for the identification of small molecule inhibitors of gsp. Plos One 9(3): E90766, 2015

Identification of small molecule inhibitors of p27Kip1 ubiquitination by high-throughput screening. 2013

Identification of novel small molecule inhibitors of adenovirus gene transfer using a high throughput screening approach. Journal of Controlled Release 170(1): 132-140, 2014

High-throughput screening assay for identification of small molecule inhibitors of Aurora2/STK15 kinase. Journal of Biomolecular Screening 9(5): 391-397, 2004

Identification of novel small-molecule inhibitors of glioblastoma cell growth and invasion by high-throughput screening. Bioscience Trends 6(4): 192-200, 2013

High-throughput screening for identification of small molecule inhibitors of histone acetyltransferases using scintillating microplates (FlashPlate). Analytical Biochemistry 298(1): 62-68, 2001

High-throughput screening assays to discover small-molecule inhibitors of protein interactions. Current Drug Discovery Technologies 5(3): 190-199, 2008

High-Throughput Screening Assays to Discover Small-Molecule Inhibitors of Protein Interactions. Current Drug Discovery Technologies 5(3): 190-199, 2008

Identification and design of novel small molecule inhibitors against MERS-CoV papain-like protease via high-throughput screening and molecular modeling. Bioorganic and Medicinal Chemistry 2019, 2019

Identification of novel small-molecule inhibitors for human transketolase by high-throughput screening with fluorescent intensity (FLINT) assay. Journal of Biomolecular Screening 9(5): 427-433, 2004

Identification of small molecule inhibitors of beta-amyloid cytotoxicity through a cell-based high-throughput screening platform. Journal of Biomolecular Screening 13(9): 870-878, 2008

High-throughput screening AlphaScreen assay for identification of small-molecule inhibitors of ubiquitin E3 ligase SCFSkp2-Cks1. Journal of Biomolecular Screening 18(8): 910-920, 2014

Ultra-High-Throughput Structure-Based Virtual Screening for Small-Molecule Inhibitors of Protein-Protein Interactions. Journal of Chemical Information and Modeling 56(2): 399-411, 2016