title: Development of a time-resolved fluorescence resonance energy transfer assay for cyclin-dependent kinase 4 and identification of its ATP-noncompetitive inhibitors.
abstract: Protein kinases are recognized as important drug targets due to the pivotal roles they play in human disease. Many kinase inhibitors are ATP competitive, leading to potential problems with poor selectivity and significant loss of potency in vivo due to cellular ATP concentrations being much higher than K(m). Consequently, there has been growing interest in the development of ATP-noncompetitive inhibitors to overcome these problems. There are challenges to identifying ATP-noncompetitive inhibitors from compound library screens because ATP-noncompetitive inhibitors are often weaker and commonly excluded by potency-based hit selection criteria in favor of abundant and highly potent ATP-competitive inhibitors in screening libraries. Here we report the development of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay for protein kinase cyclin-dependent kinase 4 (CDK4) and the identification of ATP-noncompetitive inhibitors by high-throughput screening after employing a strategy to favor this type of inhibitors. We also present kinetic characterization that is consistent with the proposed mode of inhibition.
authors: Lead Discovery, Amgen, South San Francisco, CA 94080, USA. meil@amgen.com
authors: Mei-Chu
authors: MC
authors: Lo
authors: Rachel
authors: R
authors: Ngo
authors: Kang
authors: K
authors: Dai
authors: Cong
authors: C
authors: Li
authors: Lingming
authors: L
authors: Liang
authors: Josie
authors: J
authors: Lee
authors: Renee
authors: R
authors: Emkey
authors: John
authors: J
authors: Eksterowicz
authors: Manuel
authors: M
authors: Ventura
authors: Stephen W
authors: SW
authors: Young
authors: Shou-Hua
authors: SH
authors: Xiao
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D051358:Cyclin-Dependent Kinase 4 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D008958:Models, Molecular; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*
chemical_list: D047428:Protein Kinase Inhibitors; D000255:Adenosine Triphosphate; D051358:Cyclin-Dependent Kinase 4