Multiplexed random peptide library and phospho-specific antibodies facilitate human polo-like kinase 1 inhibitor screen Journal Article

Authors: Tanaka, K.; Koresawa, M.; Iida, M.; Fukasawa, K.; Stec, E.; Cassaday, J.; Chase, P.; Rickert, K.; Hodder, P.; Takagi, T.; Komatani, H.
Article Title: Multiplexed random peptide library and phospho-specific antibodies facilitate human polo-like kinase 1 inhibitor screen
Abstract: One of the challenges to develop time-resolved fluorescence resonance energy transfer (TR-FRET) assay for serine/threonine (Ser/Thr) protein kinase is to select an optimal peptide substrate and a specific phosphor Ser/Thr antibody. This report describes a multiplexed random screen-based development of TR-FRET assay for ultra-high-throughput screening (uHTS) of small molecule inhibitors for a potent cancer drug target polo-like kinase 1 (Plk1). A screen of a diverse peptide library in a 384-well plate format identified several highly potent substrates that share the consensus motif for phosphorylation by Plk1. Their potencies were comparable to FKD peptide, a designed peptide substrate derived from well-described Plk1 substrate Cdc25C. A specific anti-phosphor Ser/Thr antibody p(S/T)F antibody that detects the phosphorylation of FKD peptide was screened out of 87 antibodies with time-resolved fluorometry technology in a 96-well plate format. Using FKD peptide and p(S/T)F antibody, we successfully developed a robust TR-FRET assay in 384-well plate format, and further miniaturized this assay to 1,536-well plate format to perform uHTS. We screened about 1.2 million compounds for Plk1 inhibitors using a Plk1 deletion mutant that only has the kinase domain and subsequently screened the same compound library using a full-length active-mutant Plk1. These uHTSs identified a number of hit compounds, and some of them had selectivity to either the deletion mutant or the full-length protein. Our results prove that a combination of random screen for substrate peptide and phospho-specific antibodies is very powerful strategy to develop TR-FRET assays for protein kinases. © Mary Ann Liebert, Inc. 2010.
Keywords: controlled study; protein phosphorylation; proto-oncogene proteins; clinical trial; protein analysis; cell cycle proteins; controlled clinical trial; randomized controlled trial; high throughput screening; enzyme activity; drug evaluation, preclinical; enzyme substrate; protein serine threonine kinase; phosphorylation; amino acid sequence; molecular sequence data; protein-serine-threonine kinases; fluorescence resonance energy transfer; antibody detection; polo like kinase 2; peptide library; fluorometry
Journal Title: Assay and Drug Development Technologies
Volume: 8
Issue: 1
ISSN: 1540-658X
Publisher: Mary Ann Liebert, Inc  
Date Published: 2010-02-01
Start Page: 47
End Page: 62
Language: English
DOI: 10.1089/adt.2009.0212
PUBMED: 20085455
PROVIDER: scopus
PMCID: PMC3532019
Notes: --- - "Export Date: 20 April 2011" - "CODEN: ADDTA" - "Source: Scopus"
Citation Impact
MSK Authors
  1. Toshimitsu Takagi
    6 Takagi