RESUMEN
The discovery of a novel series of pyrrolopyrazines as JAK inhibitors with comparable enzyme and cellular activity to tofacitinib is described. The series was identified using a scaffold hopping approach aided by structure based drug design using principles of intramolecular hydrogen bonding for conformational restriction and targeting specific pockets for modulating kinase activity.
Asunto(s)
Janus Quinasa 3/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Pirazinas/química , Pirroles/química , Diseño de Fármacos , Humanos , Janus Quinasa 3/metabolismo , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Fosforilación , Piperidinas/farmacología , Pirimidinas/farmacología , Pirroles/farmacología , Relación Estructura-ActividadRESUMEN
Using a structure based design approach we have identified a series of indazole substituted pyrrolopyrazines, which are potent inhibitors of JAK3. Intramolecular electronic repulsion was used as a strategy to induce a strong conformational bias within the ligand. Compounds bearing this conformation participated in a favorable hydrophobic interaction with a cysteine residue in the JAK3 binding pocket, which imparted high selectivity versus the kinome and improved selectivity within the JAK family.
Asunto(s)
Diseño de Fármacos , Janus Quinasa 3/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Sitios de Unión , Cristalografía por Rayos X , Interacciones Hidrofóbicas e Hidrofílicas , Indazoles/química , Janus Quinasa 1/antagonistas & inhibidores , Janus Quinasa 1/metabolismo , Janus Quinasa 2/antagonistas & inhibidores , Janus Quinasa 2/metabolismo , Janus Quinasa 3/metabolismo , Simulación del Acoplamiento Molecular , Unión Proteica , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/metabolismo , Estructura Terciaria de Proteína , Pirazinas/síntesis química , Pirazinas/química , Pirazinas/metabolismo , Relación Estructura-ActividadRESUMEN
The p53 tumor suppressor is a potent transcription factor that plays a key role in the regulation of cellular responses to stress. It is controlled by its negative regulator MDM2, which binds directly to p53 and inhibits its transcriptional activity. MDM2 also targets p53 for degradation by the proteasome. Many tumors produce high levels of MDM2, thereby impairing p53 function. Restoration of p53 activity by inhibiting the p53-MDM2 interaction may represent a novel approach to cancer treatment. RG7112 (2g) is the first clinical small-molecule MDM2 inhibitor designed to occupy the p53-binding pocket of MDM2. In cancer cells expressing wild-type p53, RG7112 stabilizes p53 and activates the p53 pathway, leading to cell cycle arrest, apoptosis, and inhibition or regression of human tumor xenografts.
RESUMEN
Activation of p53 tumor suppressor by antagonizing its negative regulator murine double minute (MDM)2 has been considered an attractive strategy for cancer therapy and several classes of p53-MDM2 binding inhibitors have been developed. However, these compounds do not inhibit the p53-MDMX interaction, and their effectiveness can be compromised in tumors overexpressing MDMX. Here, we identify small molecules that potently block p53 binding with both MDM2 and MDMX by inhibitor-driven homo- and/or heterodimerization of MDM2 and MDMX proteins. Structural studies revealed that the inhibitors bind into and occlude the p53 pockets of MDM2 and MDMX by inducing the formation of dimeric protein complexes kept together by a dimeric small-molecule core. This mode of action effectively stabilized p53 and activated p53 signaling in cancer cells, leading to cell cycle arrest and apoptosis. Dual MDM2/MDMX antagonists restored p53 apoptotic activity in the presence of high levels of MDMX and may offer a more effective therapeutic modality for MDMX-overexpressing cancers.
Asunto(s)
Apoptosis/fisiología , Hidantoínas/farmacología , Modelos Moleculares , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/metabolismo , Apoptosis/efectos de los fármacos , Western Blotting , Proteínas de Ciclo Celular , Línea Celular Tumoral , Cristalización , Dimerización , Transferencia Resonante de Energía de Fluorescencia , Humanos , Resonancia Magnética Nuclear Biomolecular , Proteínas Nucleares/química , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas c-mdm2/química , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Sales de Tetrazolio , TiazolesRESUMEN
The cyclin-dependent protein kinases are key regulators of cell cycle progression. Aberrant expression or altered activity of distinct cyclin-dependent kinase (CDK) complexes results in escape of cells from cell cycle control, leading to unrestricted cell proliferation. CDK inhibitors have the potential to induce cell cycle arrest and apoptosis in cancer cells, and identifying small-molecule CDK inhibitors has been a major focus in cancer research. Several CDK inhibitors are entering the clinic, the most recent being selective CDK2 and CDK4 inhibitors. We have identified a diaminopyrimidine compound, R547, which is a potent and selective ATP-competitive CDK inhibitor. In cell-free assays, R547 effectively inhibited CDK1/cyclin B, CDK2/cyclin E, and CDK4/cyclin D1 (K(i) = 1-3 nmol/L) and was inactive (K(i) > 5,000 nmol/L) against a panel of >120 unrelated kinases. In vitro, R547 effectively inhibited the proliferation of tumor cell lines independent of multidrug resistant status, histologic type, retinoblastoma protein, or p53 status, with IC(50)s = 0.60 mumol/L. The growth-inhibitory activity is characterized by a cell cycle block at G(1) and G(2) phases and induction of apoptosis. R547 reduced phosphorylation of the cellular retinoblastoma protein at specific CDK phosphorylation sites at the same concentrations that induced cell cycle arrest, suggesting a potential pharmacodynamic marker for clinical use. In vivo, R547 showed antitumor activity in all of the models tested to date, including six human tumor xenografts and an orthotopic syngeneic rat model. R547 was efficacious with daily oral dosing as well as with once weekly i.v. dosing in established human tumor models and at the targeted efficacious exposures inhibited phosphorylation of the retinoblastoma protein in the tumors. The selective kinase inhibition profile and the preclinical antitumor activity of R547 suggest that it may be promising for development for use in the treatment of solid tumors. R547 is currently being evaluated in phase I clinical trials.
Asunto(s)
Antineoplásicos/farmacología , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Pirimidinas/farmacología , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Apoptosis , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos Clínicos Fase I como Asunto , Quinasas Ciclina-Dependientes/metabolismo , Femenino , Fase G1/efectos de los fármacos , Fase G2/efectos de los fármacos , Genes MDR/efectos de los fármacos , Humanos , Ratones , Ratones Desnudos , Fosforilación/efectos de los fármacos , Pirimidinas/farmacocinética , Pirimidinas/uso terapéutico , Ratas , Ratas Endogámicas F344 , Retinoblastoma/tratamiento farmacológico , Retinoblastoma/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The cyclin-dependent kinases (CDKs) and their cyclin partners are key regulators of the cell cycle. Since deregulation of CDKs is found with high frequency in many human cancer cells, pharmacological inhibition of CDKs with small molecules has the potential to provide an effective strategy for the treatment of cancer. The 2,4-diamino-5-ketopyrimidines 6 reported here represent a novel class of potent and ATP-competitive inhibitors that selectively target the cyclin-dependent kinase family. This diaminopyrimidine core with a substituted 4-piperidine moiety on the C2-amino position and 2-methoxybenzoyl at the C5 position has been identified as the critical structure responsible for the CDK inhibitory activity. Further optimization has led to a good number of analogues that show potent inhibitory activities against CDK1, CDK2, and CDK4 but are inactive against a large panel of serine/threonine and tyrosine kinases (K(i) > 10 microM). As one of these representative analogues, compound 39 (R547) has the best CDK inhibitory activities (K(i) = 0.001, 0.003, and 0.001 microM for CDK1, CDK2, and CDK4, respectively) and excellent in vitro cellular potency, inhibiting the growth of various human tumor cell lines including an HCT116 cell line (IC(50) = 0.08 microM). An X-ray crystal structure of 39 bound to CDK2 has been determined in this study, revealing a binding mode that is consistent with our SAR. Compound 39 demonstrates significant in vivo efficacy in the HCT116 human colorectal tumor xenograft model in nude mice with up to 95% tumor growth inhibition. On the basis of its superior overall profile, 39 was chosen for further evaluation and has progressed into Phase I clinical trial for the treatment of cancer.