RESUMEN
A series of isoxazolo[3,4-b]quinoline-3,4(1H,9H)-diones were synthesized as potent inhibitors against Pim-1 and Pim-2 kinases. The structure-activity-relationship studies started from a high-throughput screening hit and was guided by molecular modeling of inhibitors in the active site of Pim-1 kinase. Installing a hydroxyl group on the benzene ring of the core has the potential to form a key hydrogen bond interaction to the hinge region of the binding pocket and thus resulted in the most potent inhibitor, 19, with K(i) values at 2.5 and 43.5 nM against Pim-1 and Pim-2, respectively. Compound 19 also exhibited an activity profile with a high degree of kinase selectivity.
Asunto(s)
Isoxazoles/síntesis química , Isoxazoles/farmacología , Modelos Moleculares , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Quinolinas/síntesis química , Quinolinas/farmacología , Técnicas Químicas Combinatorias , Cristalografía por Rayos X , Humanos , Isoxazoles/química , Conformación Molecular , Estructura Molecular , Quinolinas/química , Relación Estructura-ActividadRESUMEN
The synthesis and structure-activity relationships (SAR) of Chk1 inhibitors based on a 5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one core are described. Specifically, an exploration of the 7 and 8 positions on this previously disclosed core afforded compounds with improved enzymatic and cellular potency.
Asunto(s)
Antineoplásicos/farmacología , Benzodiazepinonas/farmacología , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Proteínas Quinasas/metabolismo , Antineoplásicos/síntesis química , Benzodiazepinonas/síntesis química , Línea Celular Tumoral/efectos de los fármacos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Inhibidores Enzimáticos/síntesis química , Células HeLa , Humanos , Modelos Químicos , Unión Proteica , Relación Estructura-ActividadRESUMEN
A novel series of 5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-ones have been synthesized as potent and selective checkpoint kinase 1 (Chk1) inhibitors via structure-based design. Aided by protein X-ray crystallography, medicinal chemistry efforts led to the identification of compound 46d, with potent enzymatic activity against Chk1 kinase. While maintaining a low cytotoxicity of its own, compound 46d exhibited a strong ability to abrogate G2 arrest and increased the cytotoxicity of camptothecin by 19-fold against SW620 cells. Pharmacokinetic studies revealed that it had a moderate bioavailabilty of 20% in mice. Two important binding interactions between compound 46b and Chk1 kinase, revealed by X-ray cocrystal structure, were hydrogen bonds between the hinge region and the amide bond of the core structure and a hydrogen bond between the methoxy group and Lys38 of the protein.
Asunto(s)
Antineoplásicos/síntesis química , Azepinas/síntesis química , Benzodiazepinonas/síntesis química , Inhibidores de Proteínas Quinasas/síntesis química , Proteínas Quinasas/metabolismo , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Azepinas/química , Azepinas/farmacología , Benzodiazepinonas/química , Benzodiazepinonas/farmacología , Disponibilidad Biológica , Camptotecina/farmacología , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Cristalografía por Rayos X , Doxorrubicina/farmacología , Diseño de Fármacos , Sinergismo Farmacológico , Humanos , Ratones , Modelos Moleculares , Unión Proteica , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/química , Relación Estructura-ActividadRESUMEN
The synthesis of a novel series of 1,4-dihydroindeno[1,2-c]pyrazoles with acetylene-type side chains is described. Optimization of those compounds as KDR kinase inhibitors identified 8, which displayed an oral activity in an estradiol-induced murine uterine edema model (ED50 = 3 mg/kg) superior to Sutent (ED50 = 9 mg/kg) and showed potent antitumor efficacy in an MX-1 human breast carcinoma xenograft tumor growth model (tumor growth inhibition = 90% at 25 mg/kg.day po). The compound was docked into a homology model of the homo-tetrameric pore domain of the hERG potassium channel to identify strategies to improve its cardiac safety profile. Systematic interruption of key binding interactions between 8 and Phe656, Tyr652, and Ser624 yielded 90, which only showed an IC50 of 11.6 microM in the hERG patch clamp assay. The selectivity profile for 8 and 90 revealed that both compounds are multitargeted receptor tyrosine kinase inhibitors with low nanomolar potencies against the members of the VEGFR and PDGFR kinase subfamilies.
Asunto(s)
Alquinos/síntesis química , Antineoplásicos/síntesis química , Canales de Potasio Éter-A-Go-Go/efectos de los fármacos , Indenos/síntesis química , Pirazoles/síntesis química , Receptores del Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Tiofenos/síntesis química , Alquinos/efectos adversos , Alquinos/farmacología , Animales , Antineoplásicos/efectos adversos , Antineoplásicos/farmacología , Unión Competitiva , Línea Celular , Canal de Potasio ERG1 , Edema/inducido químicamente , Edema/tratamiento farmacológico , Estradiol , Canales de Potasio Éter-A-Go-Go/fisiología , Femenino , Humanos , Indenos/efectos adversos , Indenos/farmacología , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Técnicas de Placa-Clamp , Unión Proteica , Pirazoles/efectos adversos , Pirazoles/metabolismo , Pirazoles/farmacología , Ensayo de Unión Radioligante , Estereoisomerismo , Relación Estructura-Actividad , Tiofenos/metabolismo , Tiofenos/farmacología , Enfermedades Uterinas/inducido químicamente , Enfermedades Uterinas/tratamiento farmacológico , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Mammalian cells initiate cell cycle arrest at different phases of the cell cycle in response to various forms of genotoxic stress to allow time for DNA repair, and thus preserving their genomic integrity. The protein kinases checkpoint kinase 1 (Chk1), checkpoint kinase 2 (Chk2), and mitogen-activated protein kinase-activated protein kinase 2 (MK2) have all been shown to be involved in cell cycle checkpoint control. Recently, cell cycle checkpoint abrogation has been proposed as one way to sensitize cancer cells to DNA-damaging agents due to the expected induction of mitotic catastrophe. Due to their overlapping substrate spectra and redundant functions, it is still not clear which kinase is mainly responsible for the cell cycle arrests conferred by clinically relevant chemotherapeutics. Thus, the issue remains about which kinase is the most therapeutically relevant target and, more importantly, whether multiple kinases might need to be targeted to achieve the best efficacy in light of recent studies showing superior efficacy for pan-receptor tyrosine kinase inhibitors. To clarify this issue, we investigated the roles of the three kinases in response to different genotoxic stresses through small interfering RNA-mediated specific target knockdowns. Our result showed that only the down-regulation of Chk1, but not of Chk2 or MK2, abrogated camptothecin- or 5-fluorouracil-induced S-phase arrest or doxorubicin-induced G(2)-phase arrest. This was followed by mitotic catastrophe and apoptosis. Moreover, double inhibition of Chk1 and Chk2 failed to achieve better efficacy than Chk1 inhibition alone; surprisingly, inhibition of MK2, in addition to Chk1 suppression, partially reversed the checkpoint abrogation and negated mitotic catastrophe. We further showed that this is due to the fact that in MK2-deficient cells, Cdc25A protein, which is critically required for the mitotic progression following checkpoint abrogation, becomes greatly depleted. In summary, our findings show that Chk1 is the only relevant checkpoint kinase as a cancer drug target and inhibition of other checkpoint kinases in addition to Chk1 would be nonproductive.
Asunto(s)
Ciclo Celular/fisiología , Daño del ADN/fisiología , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Antineoplásicos/farmacología , Camptotecina/farmacología , Ciclo Celular/efectos de los fármacos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Quinasa de Punto de Control 2 , Doxorrubicina/farmacología , Femenino , Fluorouracilo/farmacología , Células HeLa , Histonas/efectos de los fármacos , Histonas/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Neoplasias/tratamiento farmacológico , Proteínas Quinasas/efectos de los fármacos , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/genética , ARN Interferente Pequeño , Células Tumorales Cultivadas , Fosfatasas cdc25/efectos de los fármacos , Fosfatasas cdc25/metabolismoRESUMEN
Chk1 is the major mediator in the activation of cell-cycle checkpoints in response to a variety of genotoxic stresses. We have previously shown that inhibition of Chk1 sensitizes tumor cells to topoisomerase inhibitors such as camptothecin and doxorubicin through abrogation of cell-cycle arrest (S or G2/M checkpoints). However, it was not clear whether inhibition of Chk1 could potentiate antimetabolites, a mainstay of cancer therapy, which confer genotoxic stress through a different mechanism than topoisomerase inhibitors. 5-Fluorouracil (5-FU) is the most widely used antimetabolite in the treatment of colorectal, breast and other major types of cancers. Here we demonstrate that 5-FU activates Chk1 and induces an early S-phase arrest. Chk1 downregulation abrogates this arrest and dramatically sensitizes tumor cells to the cytotoxic effects of 5-FU. 5-FU confers S-phase arrest through Chk1-mediated Cdc25A proteolysis leading to inhibition of Cdk2. Chk1 elimination stabilizes the Cdc25A protein and results in the abrogation of the S checkpoint and resumption of DNA synthesis, which leads to excessive accumulation of double-stranded DNA breaks. As a result, downregulation of Chk1 potentiates 5-FU efficacy through induction of premature chromosomal condensation followed by apoptosis. Interestingly, the profiles of various cell-cycle markers indicate that cells progress to early M phase to induce apoptosis after checkpoint abrogation. Yet, cells fail to increase their DNA content to 4N as revealed by FACS analysis, probably due to the dramatic induction of double-stranded DNA breaks and chromosomal fragmentation. This is significantly different from the cell-cycle profiles observed in the potentiation of topoisomerase inhibitors by Chk1 siRNA, which showed mitotic progression with 4N DNA content leading to mitotic catastrophe after abrogation of the S or G2 checkpoint. Thus, our results illustrate a novel mode of checkpoint abrogation and cell death conferred by Chk1 inhibition. Additionally, we show that Chk1 deficiency potentiates 5-FU efficacy through the preferential induction of the caspase-8 pathway and subsequent caspase-3 activation. In conclusion, we have clearly demonstrated that inhibition of Chk1 not only potentiates the toxicity of conventional DNA-damaging agents such as ionizing radiation and topoisomerase inhibitors, but also enhances the toxicity of antimetabolites in cancer cell lines. This discovery reveals novel scope of checkpoint abrogation and will significantly broaden the potential application of Chk1 inhibitors in cancer therapy if they do not potentiate the toxicity of 5-FU in normal cells.
Asunto(s)
Antimetabolitos Antineoplásicos/toxicidad , Daño del ADN , Regulación hacia Abajo , Fluorouracilo/toxicidad , Proteínas Quinasas/fisiología , Apoptosis/fisiología , Caspasa 3 , Caspasa 8 , Caspasas/fisiología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Núcleo Celular/química , Núcleo Celular/efectos de los fármacos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , ADN/análisis , ADN/efectos de los fármacos , Replicación del ADN/fisiología , Resistencia a Antineoplásicos , Células HeLa , Humanos , Fosforilación , Poli(ADP-Ribosa) Polimerasas/fisiología , Proteínas Quinasas/genética , ARN Interferente Pequeño/genéticaRESUMEN
Pim-1, Pim-2, and Pim-3 are a family of serine/threonine kinases which have been found to be overexpressed in a variety of hematopoietic malignancies and solid tumors. Benzothienopyrimidinones were discovered as a novel class of Pim inhibitors that potently inhibit all three Pim kinases with subnanomolar to low single-digit nanomolar K(i) values and exhibit excellent selectivity against a panel of diverse kinases. Protein crystal structures of the bound Pim-1 complexes of benzothienopyrimidinones 3b (PDB code 3JYA), 6e (PDB code 3JYO), and 12b (PDB code 3JXW) were determined and used to guide SAR studies. Multiple compounds exhibited potent antiproliferative activity in K562 and MV4-11 cells with submicromolar EC(50) values. For example, compound 14j inhibited the growth of K562 cells with an EC(50) value of 1.7 muM and showed K(i) values of 2, 3, and 0.5 nM against Pim-1, Pim-2, and Pim-3, respectively. These novel Pim kinase inhibitors efficiently interrupted the phosphorylation of Bad in both K562 and LnCaP-Bad cell lines, indicating that their potent biological activities are mechanism-based. The pharmacokinetics of 14j was studied in CD-1 mice and shown to exhibit bioavailability of 76% after oral dosing. ADME profiling of 14j suggested a long half-life in both human and mouse liver microsomes, good permeability, modest protein binding, and no CYP inhibition below 20 muM concentration.
Asunto(s)
Antineoplásicos/síntesis química , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Pirimidinas/síntesis química , Pirimidinonas/síntesis química , Tiofenos/síntesis química , Administración Oral , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Disponibilidad Biológica , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Humanos , Técnicas In Vitro , Ratones , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Fosforilación , Conformación Proteica , Proteínas Proto-Oncogénicas c-pim-1/química , Pirimidinas/farmacocinética , Pirimidinas/farmacología , Pirimidinonas/farmacocinética , Pirimidinonas/farmacología , Relación Estructura-Actividad , Tiofenos/farmacocinética , Tiofenos/farmacología , Proteína Letal Asociada a bcl/metabolismoRESUMEN
Chk1 is the major mediator of cell-cycle checkpoints in response to various forms of genotoxic stress. Although it was previously speculated that checkpoint abrogation due to Chk1 inhibition may potentiate the efficacy of DNA-damaging agents through induction of mitotic catastrophe, there has not been direct evidence proving this process. Here, through both molecular marker and morphological analysis, we directly demonstrate that specific downregulation of Chk1 expression by Chk1 siRNA potentiates the cytotoxicities of topoisomerase inhibitors through the induction of premature chromosomal condensation and mitotic catastrophe. More importantly, we discovered that the cellular cyclin B1 level is the major determinant of the potentiation. We show that downregulation of cyclin B1 leads to impairment of the induction of mitotic catastrophe and correspondingly a reduction of the potentiation ability of either Chk1 siRNA or a small molecule Chk1 inhibitor. More significantly, we have extended the study by examining a panel of 10 cancer cell-lines with different tissue origins for their endogenous levels of cyclin B1 and the ability of a Chk1 inhibitor to sensitize the cells to DNA-damaging agents. The cellular levels of cyclin B1 positively correlate with the degrees of potentiation achieved. Of additional interest, we observed that the various colon cancer cell lines in general appear to express higher levels of cyclin B1 and also display higher sensitivity to Chk1 inhibitors, implying that Chk1 inhibitor may be more efficacious in treating colon cancers. In summary, we propose that cyclin B1 is a biomarker predictive of the efficacy of Chk1 inhibitors across different types of cancers. Unlike previously established efficacy-predictive biomarkers that are usually the direct targets of the therapeutic agents, cyclin B1 represents a non-drug-target biomarker that is based on the mechanism of action of the target inhibitor. This finding may be potentially very useful for the stratification of patients for Chk1 inhibitor clinical trials and hence, maximize its chance of success.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Ciclina B/metabolismo , Terapia Genética/métodos , Neoplasias/terapia , Proteínas Quinasas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Inhibidores de Topoisomerasa , Animales , Apoptosis/efectos de los fármacos , Biomarcadores/metabolismo , Proliferación Celular/efectos de los fármacos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Ciclina B1 , ADN-Topoisomerasas/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/administración & dosificación , Femenino , Células HeLa , Histonas/metabolismo , Humanos , Masculino , Ratones , Ratones SCID , Mitosis/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Neoplasias/genética , Neoplasias/patología , Fosforilación , Inhibidores de Proteínas Quinasas/administración & dosificación , Proteínas Quinasas/genética , Factores de Tiempo , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We report the synthesis and biological evaluation of 5-substituted 1,4-dihydroindeno[1,2-c]pyrazoles as multitargeted kinase inhibitors. Initial efforts focused on the development of selective KDR inhibitors, while later strategies involved the improvement of potency toward multiple kinase targets. Thus, several compounds were identified as potent KDR, Flt1, Flt3, and c-Kit inhibitors.
Asunto(s)
Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/química , Pirazoles/farmacología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Animales , Humanos , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Pirazoles/síntesis químicaRESUMEN
A new series of potent tricyclic pyrazole-based Chk1 inhibitors are described. Analogues disubstituted on the 6- and 7-positions show improved Chk1 inhibition potency compared with analogues with a single substituent on either the 6- or 7-position. Based on the lead compound 4'-(6,7-dimethoxy-2,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-biphenyl-4-ol (2), detailed SAR studies on the 6- and 7-positions were performed. 3'-morpholin-4'-yl-propoxy, pyridin-4'-ylmethoxy, pyridin-3'-ylmethoxy, 2'-(5''-ethyl-pyridin-2''-yl)-ethoxy, pyridin-2'-ylethoxy, (6'-methyl-pyridin-2'-yl)-propoxyethoxy, 2',3'-dihydroxyl-1'-yl-propoxy, and tetrahydro-furan-3'-yloxy have been identified as the best groups on the 6-position when the 7-position is substituted with methoxyl group. Pyridin-2'-ylmethoxy and pyridin-3'-ylmethoxy have been identified as the best substituents at the 7-position while the 6-position bearing methoxyl group. These compounds significantly potentiate the cytotoxicity of DNA-damaging antitumor agents in a cell-based assay and efficiently abrogate the doxorubicin-induced G2/M and the camptothecin-induced S checkpoints, suggesting that their potent biological activities are mechanism-based through Chk1 inhibition.
Asunto(s)
Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/efectos de los fármacos , Pirazoles/síntesis química , Pirazoles/farmacología , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Evaluación Preclínica de Medicamentos , Células HeLa , Humanos , Inhibidores de Proteínas Quinasas/química , Pirazoles/químicaRESUMEN
An extensive structure-activity relationship study of the 3-position of a series of tricyclic pyrazole-based Chk1 inhibitors is described. As a result, 4'-(1,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-benzonitriles (4) and 4'-(1,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-pyridine-2'-carbonitriles (29) emerged as new lead series. Compared with the original lead compound 2, these new leads fully retain the biological activity in both enzymatic inhibition and cell-based assays. More importantly, the new leads 4 and 29 exhibit favorable physicochemical properties such as lower molecular weight, lower Clog P, and the absence of a hydroxyl group. Furthermore, structure-activity relationship studies were performed at the 6- and 7-positions of 4, which led to the identification of ideal Chk1 inhibitors 49, 50, 51, and 55. These compounds not only potently inhibit Chk1 in an enzymatic assay but also significantly potentiate the cytotoxicity of DNA-damaging agents in cell-based assays while they show little single agent activity. A cell cycle analysis by FACS confirmed that these Chk1 inhibitors efficiently abrogate the G2/M and S checkpoints induced by DNA-damaging agent. The current work paved the way to the identification of several potent Chk1 inhibitors with good pharmacokinetics that are suitable for in vivo study with oral dosing.
Asunto(s)
Nitrilos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/efectos de los fármacos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Nitrilos/química , Inhibidores de Proteínas Quinasas/química , Relación Estructura-ActividadRESUMEN
A new class of checkpoint kinase 1 (CHK-1) inhibitors bearing a 1,4-dihydroindeno[1,2-c]pyrazole core was developed after initial hits from high throughput screening. The efficient hit-to-lead process was facilitated by X-ray crystallography and led to potent inhibitors (<10nM) against CHK-1. X-ray co-crystal structures of bound inhibitors demonstrated that two sub-series of this class of compounds, exemplified by 21 and 41, exhibit distinctive hydrogen bonding patterns in the specificity pocket of the active site. Two compounds, 41 and 43, were capable of potentiating doxorubicin and camptothecin, both DNA-damaging agents, in cell proliferation assays (MTS and soft agar assays) and abrogating G2/M checkpoint in a mechanism-based FACS assay.
Asunto(s)
Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Pirazoles/síntesis química , Pirazoles/farmacología , Antineoplásicos Fitogénicos/farmacología , Camptotecina/farmacología , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Sinergismo Farmacológico , Células HeLa , Humanos , Enlace de Hidrógeno , Indicadores y Reactivos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Especificidad por SustratoRESUMEN
A series of 1,4-dihydroindeno[1,2-c]pyrazole compounds with a cyanopyridine moiety at the 3-position of the tricyclic pyrazole core was explored as potent CHK-1 inhibitors. The impact of substitutions at the 6 and/or 7-position of the core on pharmacokinetic properties was studied in detail. Compounds carrying a side chain with an ether linker at the 7-position and a terminal morpholino group, such as 29 and 30, exhibited much-improved oral biovailability in mice as compared to earlier generation inhibitors. These compounds also possessed desirable cellular activity in potentiating doxorubicin and will serve as valuable tool compounds for in vivo evaluation of CHK-1 inhibitors to sensitize DNA-damaging agents.
Asunto(s)
Hidrógeno/química , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Pirazoles/química , Pirazoles/farmacología , Piridinas/química , Administración Oral , Animales , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Cianuros/química , Indenos/química , Concentración 50 Inhibidora , Ratones , Estructura Molecular , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/síntesis química , Pirazoles/administración & dosificación , Pirazoles/síntesis química , Ratas , Relación Estructura-ActividadRESUMEN
A study on substitutions at the four open positions on the phenyl ring of the 1,4-dihydroindeno[1,2-c]pyrazoles as potent CHK-1 inhibitors is described. Bis-substitution at both the 6- and 7-positions led to inhibitors with IC(50) values below 0.3nM. The compound with the best overall activities (36) was able to potentiate the anti-proliferative effect of doxorubicin in HeLa cells by at least 47-fold. Physicochemical, metabolic, and pharmacokinetic properties of selected inhibitors are also disclosed.
Asunto(s)
Antineoplásicos/farmacocinética , Química Farmacéutica/métodos , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores de Proteínas Quinasas/farmacocinética , Proteínas Quinasas/química , Animales , Antineoplásicos/química , Células CACO-2 , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Daño del ADN , Diseño de Fármacos , Citometría de Flujo , Humanos , Concentración 50 Inhibidora , Ratones , Microsomas Hepáticos/metabolismo , Inhibidores de Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , RatasRESUMEN
A series of 1,4-dihydroindeno[1,2-c]pyrazoles with a 3-thiophene substituent carrying a urea-type side chain were identified as potent multitargeted (VEGFR and PDGFR families) receptor tyrosine kinase inhibitors. A KDR homology model suggested that the urea moiety is able to interact with a recognition motif in the hydrophobic specificity pocket of the enzyme.
Asunto(s)
Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirazoles/síntesis química , Pirazoles/farmacología , Secuencias de Aminoácidos , Química Farmacéutica/métodos , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Enlace de Hidrógeno , Concentración 50 Inhibidora , Microsomas Hepáticos/metabolismo , Modelos Químicos , Modelos Moleculares , Urea/químicaRESUMEN
A series of 1,4-dihydroindeno[1,2-c]pyrazoles was prepared and evaluated for their enzymatic inhibition of KDR kinase. Computer modeling studies revealed the importance of attaching a basic side chain in predicting the binding mode of those compounds. Further investigation of structure-activity relationships led to 19, a lead compound with an acceptable selectivity profile, activity in whole cells, and good oral efficacy in an estradiol-induced murine uterine edema model of VEGF activity.
Asunto(s)
Indenos/farmacología , Pirazoles/farmacología , Administración Oral , Animales , Química Farmacéutica , Diseño de Fármacos , Edema/patología , Estradiol/farmacología , Femenino , Indenos/química , Concentración 50 Inhibidora , Ratones , Modelos Químicos , Modelos Moleculares , Pirazoles/química , Relación Estructura-Actividad , Útero/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/químicaRESUMEN
The majority of cancer therapeutics induces DNA damage to kill cells. Normal proliferating cells undergo cell cycle arrest in response to DNA damage, thus allowing DNA repair to protect the genome. DNA damage induced cell cycle arrest depends on an evolutionarily conserved signal transduction network in which the Chk1 kinase plays a critical role. In mammalian cells, the p53 and RB pathways further augment the cell cycle arrest response to prevent catastrophic cell death. Given the fact that most tumor cells suffer defects in the p53 and RB pathways, it is likely that tumor cells would depend more on the Chk1 kinase to maintain cell cycle arrest than would normal cells. Therefore Chk1 inhibition could be used to specifically sensitize tumor cells to DNA-damaging agents. We have previously shown that siRNA-mediated Chk1 knockdown abrogates DNA damage-induced checkpoints and potentiates the cytotoxicity of several DNA-damaging agents in p53-deficient cell lines. In this study, we have developed 2 potent and selective Chk1 inhibitors, A-690002 and A-641397, and shown that these compounds abrogate cell cycle checkpoints and potentiate the cytotoxicity of topoisomerase inhibitors and gamma-radiation in p53-deficient but not in p53-proficient cells of different tissue origins. These results indicate that it is feasible to achieve a therapeutic window with 1 or more Chk1 inhibitors in potentiation of cancer therapy based on the status of the p53 pathway in a wide spectrum of tumor types.
Asunto(s)
Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Proteína p53 Supresora de Tumor/deficiencia , Urea/análogos & derivados , Anticuerpos/farmacología , Western Blotting , Proteína Quinasa CDC2/inmunología , Proteína Quinasa CDC2/metabolismo , Camptotecina/farmacología , Caspasas/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Daño del ADN , Relación Dosis-Respuesta a Droga , Doxorrubicina/farmacología , Sinergismo Farmacológico , Células HeLa , Humanos , Estructura Molecular , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/química , Proteínas Quinasas/genética , Proteínas Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Interferente Pequeño/genética , Factores de Tiempo , Proteína p53 Supresora de Tumor/genética , Urea/química , Urea/farmacología , Fosfatasas cdc25/genética , Fosfatasas cdc25/metabolismoRESUMEN
Paclitaxel (Taxol) is the most-prescribed anti-mitotic agent for a variety of advanced metastatic cancers. It induces mitotic arrest leading to apoptosis through microtubule stabilization. Chk1 is the major cell-cycle checkpoint kinase mediating S- and G2-arrests in response to various DNA-damages. Chk1 inhibitor is anticipated and has been demonstrated to potentiate the cytotoxicity of DNA-damaging agents through abrogation of cell-cycle checkpoints. Paclitaxel does not, however, induce Chk1 activation, and Chk1 has not been shown to function in mitotic checkpoint. Thus, Chk1 inhibitor is not expected to enhance the toxicity of paclitaxel. Here we show that downregulation of Chk1 sensitizes tumor cells to the toxicity of paclitaxel in cell proliferation assay. Fluorescence microscopy showed that Chk1 knockdown augments mitotic catastrophe and apoptosis in paclitaxel-treated cancer cells. Further, we elucidated the mechanism of this sensitization. Chk1 inhibition facilitates paclitaxel-induced M-phase entry by activation of Cdc2 kinase and accumulation of cyclin B1, the required cofactor for Cdc2 kinase activity. Moreover, Chk1 downregulation inhibits M phase exit through induction of the anaphase inhibitor, securin/PDS1. Collectively, Chk1 elimination sustains a more effective mitotic arrest as demonstrated by the more efficient accumulation of M-phase marker phospho-histone H3. We show that Chk1 elimination attenuates the paclitaxel-induced activation of the anti-apoptotic p42/p44 (ERK1/2) MAP kinase pathway, additionally contributing to the sensitization. Our results suggest that in addition to its well-established role as an enforcer of S and G2-checkpoints in response to genotoxic stress, Chk1 also plays a protective role in mitotic checkpoint to lessen mitotic catastrophe and thereby limits cell-death. Therefore Chk1 downregulation can not only potentiate DNA-damaging agents, but also enhance the toxicity of anti-microtubule agents, which significantly broadens its therapeutic applications.
Asunto(s)
Antineoplásicos/toxicidad , Inhibidores Enzimáticos/toxicidad , Paclitaxel/toxicidad , Proteínas Quinasas/metabolismo , ARN Interferente Pequeño/farmacología , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Neoplasias del Colon , Células HeLa , Humanos , Neoplasias Pulmonares , Mitosis/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Quinasas/genéticaRESUMEN
A short and versatile synthesis of substituted quinolines is provided. Alkylation of sodium tolylsulfinate with bromomethyl- or chloromethyl ketones generates beta-keto sulfones. Knoevenagel condensation of the beta-keto sulfones with an aldehyde provides alpha-tolylsulfonyl-alpha,beta-unsaturated ketones. Michael addition of the dianion of N-Boc-anilines in the presence of CuCN and LiCl with the unsaturated ketone generates a 1,4-adduct, which after deprotection of the Boc group and thermal elimination of the tolyl sulfone provides the quinoline.
RESUMEN
UV and ionizing radiation (IR) activate DNA damage checkpoints and induce Cdc25A degradation (Mailand, N., Falck, J., Lukas, C., Syljuasen, R. G., Welcker, M., Bartek, J., and Lukas, J. (2000) Science 288, 1425-1429; Falck, J., Mailand, N., Syljuasen, R. G., Bartek, J., and Lukas J. (2001) Nature 410, 842-847). The degradation of Cdc25A is abrogated by caffeine, which implicates Chk1 as the potential mediator (Mailand, N., Falck, J., Lukas, C., Syljuasen, R. G., Welcker, M., Bartek, J., and Lukas, J. (2000) Science 288, 1425-1429). However, the involvement of Chk1 is far from clear, because caffeine is a rather nonspecific inhibitor of the ATR/Chk1 signaling pathway. Additionally, it is not known whether DNA-damaging drugs commonly used in chemotherapy, which may activate different signal transduction pathways than UV or IR, also confer Cdc25A degradation. Herein, we show that camptothecin and doxorubicin, two widely used topoisomerase inhibitors conferring S and G2 arrest, respectively, cause the degradation of Cdc25A. Using a small interfering RNA that enables the specific elimination of Chk1 expression, we show that the observed proteolysis of Cdc25A is mediated through Chk1. Moreover, Cdc25A overexpression abrogates the Chk1-mediated degradation and overcomes the doxorubicin-induced G2 arrest through dephosphorylation and activation of Cdc2/Cdk1 in a dose-dependent manner. These results suggest that: (a) Cdc25A is involved in the G2/M transition in addition to its commonly accepted effect on G1/S progression, and (b) Chk1 mediates both S and G2 checkpoint and is thus a more ubiquitous cell cycle checkpoint mediator than previously thought.