Multiplexed random peptide library and phospho-specific antibodies facilitate human polo-like kinase 1 inhibitor screen.
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.
Tanaka
Kenji
K
Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd., Tsukuba, Japan. kenji-tanaka@taiho.co.jp
Koresawa
Mitsunori
M
Iida
Masato
M
Fukasawa
Kazuhiro
K
Stec
Erica
E
Cassaday
Jason
J
Chase
Peter
P
Rickert
Keith
K
Hodder
Peter
P
Takagi
Toshimitsu
T
Komatani
Hideya
H
D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008969:Molecular Sequence Data; D019151:Peptide Library; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry; D000097869:Polo-Like Kinase 1
D018797:Cell Cycle Proteins; D019151:Peptide Library; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D000097869:Polo-Like Kinase 1