Open AccessMultiplexed Random Peptide Library and Phospho-Specific Antibodies Facilitate Human Polo-Like Kinase 1 Inhibitor Screen
Author(s) -
Kenji Tanaka,
Mitsuhiko Koresawa,
Masato Iida,
Kazuhiro Fukasawa,
Erica Stec,
Jason Cassaday,
Peter Chase,
Keith Rickert,
Peter Hodder,
Toshimitsu Takagi,
Hideya Komatani
Publication year - 2010
Publication title -
assay and drug development technologies
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.402
H-Index - 56
eISSN - 1557-8127
pISSN - 1540-658X
DOI - 10.1089/adt.2009.0212
Subject(s) - förster resonance energy transfer , plk1 , peptide , peptide library , kinase , phosphorylation , microbiology and biotechnology , protein tag , biochemistry , chemistry , biology , peptide sequence , fluorescence , fusion protein , cell cycle , recombinant dna , physics , quantum mechanics , gene , cell
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.