Multiplexed random peptide library and phospho-specific antibodies facilitate human polo-like kinase 1 inhibitor screen.
Assay Drug Dev Technol
; 8(1): 47-62, 2010 Feb.
Article
en En
| MEDLINE
| ID: mdl-20085455
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.
Texto completo:
1
Base de datos:
MEDLINE
Asunto principal:
Proteínas Proto-Oncogénicas
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Proteínas Serina-Treonina Quinasas
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Proteínas de Ciclo Celular
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Biblioteca de Péptidos
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Evaluación Preclínica de Medicamentos
Tipo de estudio:
Clinical_trials
Idioma:
En
Revista:
Assay Drug Dev Technol
Asunto de la revista:
FARMACOLOGIA
Año:
2010
Tipo del documento:
Article