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J Biomol Screen ; 17(1): 39-48, 2012 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21859681


A high-throughput RapidFire mass spectrometry assay is described for the JMJD2 family of Fe(2+), O(2), and α-ketoglutarate-dependent histone lysine demethylases. The assay employs a short amino acid peptide substrate, corresponding to the first 15 amino acid residues of histone H3, but mutated at two positions to increase assay sensitivity. The assay monitors the direct formation of the dimethylated-Lys9 product from the trimethylated-Lys9 peptide substrate. Monitoring the formation of the monomethylated and des-methylated peptide products is also possible. The assay was validated using known inhibitors of the histone lysine demethylases, including 2,4-pyridinedicarboxylic acid and an α-ketoglutarate analogue. With a sampling rate of 7 s per well, the RapidFire technology permitted the single-concentration screening of 101 226 compounds against JMJD2C in 10 days using two instruments, typically giving Z' values of 0.75 to 0.85. Several compounds were identified of the 8-hydroxyquinoline chemotype, a known series of inhibitors of the Lys9-specific histone demethylases. The peptide also functions as a substrate for JMJD2A, JMJD2D, and JMJD2E, thus enabling the development of assays for all 3 enzymes to monitor progress in compound selectivity. The assay represents the first report of a RapidFire mass spectrometry assay for an epigenetics target.

Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/farmacología , Ensayos Analíticos de Alto Rendimiento/métodos , Histona Demetilasas/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Espectrometría de Masas/métodos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/metabolismo , Epigénesis Genética/efectos de los fármacos , Histona Demetilasas/antagonistas & inhibidores , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Cinética , Lisina/metabolismo , Oxiquinolina/metabolismo , Oxiquinolina/farmacología , Péptidos/metabolismo , Piridinas/metabolismo , Piridinas/farmacología , Especificidad por Sustrato
J Med Chem ; 52(2): 379-88, 2009 Jan 22.
Artículo en Inglés | MEDLINE | ID: mdl-19143567


Here, we describe the identification of a clinical candidate via structure-based optimization of a ligand efficient pyrazole-benzimidazole fragment. Aurora kinases play a key role in the regulation of mitosis and in recent years have become attractive targets for the treatment of cancer. X-ray crystallographic structures were generated using a novel soakable form of Aurora A and were used to drive the optimization toward potent (IC(50) approximately 3 nM) dual Aurora A/Aurora B inhibitors. These compounds inhibited growth and survival of HCT116 cells and produced the polyploid cellular phenotype typically associated with Aurora B kinase inhibition. Optimization of cellular activity and physicochemical properties ultimately led to the identification of compound 16 (AT9283). In addition to Aurora A and Aurora B, compound 16 was also found to inhibit a number of other kinases including JAK2 and Abl (T315I). This compound demonstrated in vivo efficacy in mouse xenograft models and is currently under evaluation in phase I clinical trials.

Bencimidazoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Urea/análogos & derivados , Animales , Aurora Quinasa A , Aurora Quinasa B , Aurora Quinasas , Bencimidazoles/química , Bencimidazoles/farmacocinética , Línea Celular Tumoral , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Humanos , Ratones , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Relación Estructura-Actividad , Urea/química , Urea/farmacocinética , Urea/farmacología