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1.
ACS Med Chem Lett ; 10(4): 534-538, 2019 Apr 11.
Artículo en Inglés | MEDLINE | ID: mdl-30996792

RESUMEN

Poly(ADP-ribose) polymerase-1 (PARP-1) is an enzyme involved in signaling and repair of DNA single strand breaks. PARP-1 employs NAD+ to modify substrate proteins via the attachment of poly(ADP-ribose) chains. PARP-1 is a well established target in oncology, as testified by the number of marketed drugs (e.g., Lynparza, Rubraca, Zejula, and Talzenna) used for the treatment of ovarian, breast, and prostate tumors. Efforts in investigating an uncharted region of the previously identified isoindolinone carboxamide series delivered (S)-13 (NMS-P515), a potent inhibitor of PARP-1 both in biochemical (K d: 0.016 µM) and cellular (IC50: 0.027 µM) assays. Cocrystal structure allowed explaining NMS-P515 stereospecific inhibition of the target. After having ruled out potential loss of enantiopurity in vitro and in vivo, NMS-P515 was synthesized in an asymmetric fashion. NMS-P515 ADME profile and its antitumor activity in a mouse xenograft cancer model render the compound eligible for further optimization.

2.
J Med Chem ; 58(17): 6875-98, 2015 Sep 10.
Artículo en Inglés | MEDLINE | ID: mdl-26222319

RESUMEN

The nuclear protein poly(ADP-ribose) polymerase-1 (PARP-1) has a well-established role in the signaling and repair of DNA and is a prominent target in oncology, as testified by the number of candidates in clinical testing that unselectively target both PARP-1 and its closest isoform PARP-2. The goal of our program was to find a PARP-1 selective inhibitor that would potentially mitigate toxicities arising from cross-inhibition of PARP-2. Thus, an HTS campaign on the proprietary Nerviano Medical Sciences (NMS) chemical collection, followed by SAR optimization, allowed us to discover 2-[1-(4,4-difluorocyclohexyl)piperidin-4-yl]-6-fluoro-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide (NMS-P118, 20by). NMS-P118 proved to be a potent, orally available, and highly selective PARP-1 inhibitor endowed with excellent ADME and pharmacokinetic profiles and high efficacy in vivo both as a single agent and in combination with Temozolomide in MDA-MB-436 and Capan-1 xenograft models, respectively. Cocrystal structures of 20by with both PARP-1 and PARP-2 catalytic domain proteins allowed rationalization of the observed selectivity.


Asunto(s)
Antineoplásicos/química , Isoindoles/química , Piperidinas/química , Inhibidores de Poli(ADP-Ribosa) Polimerasas/química , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Disponibilidad Biológica , Proliferación Celular/efectos de los fármacos , Dacarbazina/administración & dosificación , Dacarbazina/análogos & derivados , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Xenoinjertos , Ensayos Analíticos de Alto Rendimiento , Humanos , Isoindoles/administración & dosificación , Isoindoles/farmacología , Ratones Endogámicos BALB C , Ratones Desnudos , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Trasplante de Neoplasias , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Piperidinas/administración & dosificación , Piperidinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/administración & dosificación , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Ratas Sprague-Dawley , Relación Estructura-Actividad , Temozolomida , Neoplasias de la Mama Triple Negativas
3.
J Med Chem ; 52(14): 4380-90, 2009 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-19555113

RESUMEN

Cdc7 kinase has recently emerged as an attractive target for cancer therapy and low-molecular-weight inhibitors of Cdc7 kinase have been found to be effective in the inhibition of tumor growth in animal models. In this paper, we describe synthesis and structure-activity relationships of new 1H-pyrrolo[2,3-b]pyridine derivatives identified as inhibitors of Cdc7 kinase. Progress from (Z)-2-phenyl-5-(1H-pyrrolo[2,3-b]pyridin-3-ylmethylene)-3,5-dihydro-4H-imidazol-4-one (1) to [(Z)-2-(benzylamino)-5-(1H-pyrrolo[2,3-b]pyridin-3-ylmethylene)-1,3-thiazol-4(5H)-one] (42), a potent ATP mimetic inhibitor of Cdc7 kinase with IC(50) value of 7 nM, is also reported.


Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Piridinas/síntesis química , Piridinas/farmacología , Proteínas de Ciclo Celular/química , Línea Celular , Humanos , Modelos Moleculares , Conformación Molecular , Inhibidores de Proteínas Quinasas/análogos & derivados , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/química , Piridinas/química , Relación Estructura-Actividad
4.
Nat Chem Biol ; 4(6): 357-65, 2008 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18469809

RESUMEN

Cdc7 is an essential kinase that promotes DNA replication by activating origins of replication. Here, we characterized the potent Cdc7 inhibitor PHA-767491 (1) in biochemical and cell-based assays, and we tested its antitumor activity in rodents. We found that the compound blocks DNA synthesis and affects the phosphorylation of the replicative DNA helicase at Cdc7-dependent phosphorylation sites. Unlike current DNA synthesis inhibitors, PHA-767491 prevents the activation of replication origins but does not impede replication fork progression, and it does not trigger a sustained DNA damage response. Treatment with PHA-767491 results in apoptotic cell death in multiple cancer cell types and tumor growth inhibition in preclinical cancer models. To our knowledge, PHA-767491 is the first molecule that directly affects the mechanisms controlling initiation as opposed to elongation in DNA replication, and its activities suggest that Cdc7 kinase inhibition could be a new strategy for the development of anticancer therapeutics.


Asunto(s)
Antineoplásicos/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Replicación del ADN/efectos de los fármacos , ADN/efectos de los fármacos , Piperidonas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirroles/farmacología , Animales , Antineoplásicos/química , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , ADN/biosíntesis , Relación Dosis-Respuesta a Droga , Fibroblastos/efectos de los fármacos , Células HeLa , Humanos , Ratones , Ratones Desnudos , Ratones SCID , Componente 2 del Complejo de Mantenimiento de Minicromosoma , Estructura Molecular , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/química , Fosforilación , Piperidonas/química , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Pirroles/química , Ratas , Bibliotecas de Moléculas Pequeñas , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de Xenoinjerto
5.
J Biol Chem ; 282(1): 208-15, 2007 Jan 05.
Artículo en Inglés | MEDLINE | ID: mdl-17062569

RESUMEN

Cdc7 kinase promotes and regulates DNA replication in eukaryotic organisms. Multiple mechanisms modulating kinase activity in response to DNA replication stress have been reported, supporting the opposing notions that Cdc7 either plays an active role under these conditions or, conversely, is a final target inactivated by a checkpoint response. We have developed new immnunological reagents to study the properties of human Cdc7 kinase in cells challenged with the ribonucleotide reductase inhibitor hydroxyurea or with the DNA topoisomerase II inhibitor etoposide. We show that Cdc7.Dbf4 and Cdc7.Drf1 complexes are stable and active in multiple cell lines upon drug treatment, with Cdc7.Dbf4 accumulating on chromatin-enriched fractions. Cdc7 depletion by small interfering RNA in hydroxyurea and etoposide impairs hyper-phosphorylation of Mcm2 at specific Cdc7-dependent phosphorylation sites and drug-induced hyper-phosphorylation of chromatin-bound Mcm4. Furthermore, sustained inhibition of Cdc7 in the presence of these drugs increases cell death supporting the notion that the Cdc7 kinase plays a role in maintaining cell viability during replication stress.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Apoptosis , Proteínas de Ciclo Celular/fisiología , Supervivencia Celular , Cromatina/química , ADN/química , Etopósido/química , Etopósido/farmacología , Células HeLa , Humanos , Hidroxiurea/química , Hidroxiurea/farmacología , Componente 2 del Complejo de Mantenimiento de Minicromosoma , Proteínas Nucleares/fisiología , Fosforilación , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ribonucleótido Reductasas/antagonistas & inhibidores
6.
J Biol Chem ; 281(15): 10281-90, 2006 Apr 14.
Artículo en Inglés | MEDLINE | ID: mdl-16446360

RESUMEN

Minichromosome maintenance 2-7 proteins play a pivotal role in replication of the genome in eukaryotic organisms. Upon entry into S-phase several subunits of the MCM hexameric complex are phosphorylated. It is thought that phosphorylation activates the intrinsic MCM DNA helicase activity, thus allowing formation of active replication forks. Cdc7, Cdk2, and ataxia telangiectasia and Rad3-related kinases regulate S-phase entry and S-phase progression and are known to phosphorylate the Mcm2 subunit. In this work, by in vitro kinase reactions and mass spectrometry analysis of the products, we have mapped phosphorylation sites in the N terminus of Mcm2 by Cdc7, Cdk2, Cdk1, and CK2. We found that Cdc7 phosphorylates Mcm2 in at least three different sites, one of which corresponds to a site also reported to be phosphorylated by ataxia telangiectasia and Rad3-related. Three serine/proline sites were identified for Cdk2 and Cdk1, and a unique site was phosphorylated by CK2. We raised specific anti-phosphopeptide antibodies and found that all the sites identified in vitro are also phosphorylated in cells. Importantly, although all the Cdc7-dependent Mcm2 phosphosites fluctuate during the cell cycle with kinetics similar to Cdc7 kinase activity and Cdc7 protein levels, phosphorylation of Mcm2 in the putative cyclin-dependent kinase (Cdk) consensus sites is constant during the cell cycle. Furthermore, our analysis indicates that the majority of the Mcm2 isoforms phosphorylated by Cdc7 are not stably associated with chromatin. This study forms the basis for understanding how MCM functions are regulated by multiple kinases within the cell cycle and in response to external perturbations.


Asunto(s)
Proteínas de Ciclo Celular/fisiología , Proteínas Nucleares/fisiología , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Western Blotting , Proteína Quinasa CDC2/metabolismo , Quinasa de la Caseína II/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Cromatina/química , Cromatografía Liquida , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , ADN Helicasas/química , Electroforesis en Gel de Poliacrilamida , Fibroblastos/metabolismo , Células HeLa , Humanos , Iones , Luciferasas/metabolismo , Espectrometría de Masas , Microscopía Fluorescente , Componente 2 del Complejo de Mantenimiento de Minicromosoma , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Péptidos/química , Fosforilación , Prolina/química , Isoformas de Proteínas , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Serina/química , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Timidina/química , Transfección , Tripsina/farmacología , Quinasa Activadora de Quinasas Ciclina-Dependientes
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