RESUMEN
The inhibition of KSP causes mitotic arrest by activating the spindle assembly checkpoint. While transient inhibition of KSP leads to reversible mitotic arrest, prolonged exposure to a KSP inhibitor induces apoptosis. Induction of apoptosis by the KSP inhibitor couples with mitotic slippage. Slippage-refractory cells show resistance to KSP inhibitor-mediated lethality, whereas promotion of slippage after mitotic arrest enhances apoptosis. However, attenuation of the spindle checkpoint confers resistance to KSP inhibitor-induced apoptosis. Furthermore, sustained KSP inhibition activates the proapoptotic protein, Bax, and both activation of the spindle checkpoint and subsequent mitotic slippage are required for Bax activation. These studies indicate that in response to KSP inhibition, activation of the spindle checkpoint followed by mitotic slippage initiates apoptosis by activating Bax.
Asunto(s)
Apoptosis , Genes cdc/fisiología , Cinesinas/antagonistas & inhibidores , Mitosis/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Huso Acromático/fisiología , Caspasa 3 , Caspasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Resistencia a Medicamentos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Células HeLa , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Estructura Molecular , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Pirroles/farmacología , Proteína X Asociada a bcl-2RESUMEN
Translation of significant biochemical activity of pyridyl aminothiazole class of Chk1 inhibitors into functional CEA potency required analysis and adjustment of both physical properties and kinase selectivity profile of the series. The steps toward optimization of cellular potency included elimination of CDK7 activity, reduction of molecular weight and polar surface area and increase in lipophilicity of the molecules in the series.
Asunto(s)
Antineoplásicos/síntesis química , Inhibidores de Proteínas Quinasas/síntesis química , Proteínas Quinasas/química , Piridinas/síntesis química , Tiazoles/síntesis química , Antineoplásicos/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/química , Diseño de Fármacos , Halogenación , Humanos , Cinética , Estructura Molecular , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Piridinas/farmacología , Relación Estructura-Actividad , Tiazoles/farmacología , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
Pyridyl aminothiazoles comprise a novel class of ATP-competitive Chk1 inhibitors with excellent inhibitory potential. Modification of the core with ethylenediamine amides provides compounds with low picomolar potency and very high residence times. Investigation of binding parameters of such compounds using X-ray crystallography and molecular dynamics simulations revealed multiple hydrogen bonds to the enzyme backbone as well as stabilization of the conserved water molecules network in the hydrophobic binding region.
Asunto(s)
Antineoplásicos/síntesis química , Inhibidores de Proteínas Quinasas/síntesis química , Proteínas Quinasas/química , Piridinas/síntesis química , Tiazoles/síntesis química , Amidas/química , Antineoplásicos/farmacología , Sitios de Unión , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Cristalografía por Rayos X , Diseño de Fármacos , Etilenodiaminas/química , Humanos , Enlace de Hidrógeno , Cinética , Simulación de Dinámica Molecular , Estructura Molecular , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Piridinas/farmacología , Relación Estructura-Actividad , Tiazoles/farmacología , Agua/químicaRESUMEN
The kinesin spindle protein (KSP), a microtubule motor protein, is essential for the formation of bipolar spindles during mitosis. Inhibition of KSP activates the spindle checkpoint and causes apoptosis. It was shown that prolonged inhibition of KSP activates Bax and caspase-3, which requires a competent spindle checkpoint and couples with mitotic slippage. Here we investigated how Bax is activated by KSP inhibition and the roles of Bax and p53 in KSP inhibitor-induced apoptosis. We demonstrate that small interfering RNA-mediated knockdown of Bax greatly attenuates KSP inhibitor-induced apoptosis and that Bax activation is upstream of caspase activation. This indicates that Bax mediates the lethality of KSP inhibitors and that KSP inhibition provokes apoptosis via the intrinsic apoptotic pathway where Bax activation is prior to caspase activation. Although the BH3-only protein Puma is induced after mitotic slippage, suppression of de novo protein synthesis that abrogates Puma induction does not block activation of Bax or caspase-3, indicating that Bax activation is triggered by a posttranslational event. Comparison of KSP inhibitor-induced apoptosis between matched cell lines containing either functional or deficient p53 reveals that inhibition of KSP induces apoptosis independently of p53 and that p53 is dispensable for spindle checkpoint function. Thus, KSP inhibitors should be active in p53-deficient tumors.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/biosíntesis , Apoptosis/fisiología , Caspasa 3/metabolismo , Cinesinas/fisiología , Proteínas Proto-Oncogénicas/biosíntesis , Proteína p53 Supresora de Tumor/fisiología , Proteína X Asociada a bcl-2/biosíntesis , Antibióticos Antineoplásicos/farmacología , Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Doxorrubicina/farmacología , Humanos , Cinesinas/antagonistas & inhibidores , Paclitaxel/farmacología , Procesamiento Proteico-Postraduccional , Transducción de Señal , Huso AcromáticoRESUMEN
We describe herein a novel series of 3-amino-4-hydrazine-cyclobut-3-ene-1,2-diones as potent and selective inhibitors against the CXCR2 chemokine receptor and IL-8-mediated chemotaxis of a CXCR2-expressing cell line. Furthermore, these alkyl-hydrazine series inhibitors such as 5b demonstrated acceptable metabolic stability when incubated in human and rat microsomes.
Asunto(s)
Antiinflamatorios/síntesis química , Hidrazinas/síntesis química , Receptores de Interleucina-8B/antagonistas & inhibidores , Animales , Antiinflamatorios/química , Antiinflamatorios/farmacología , Células CHO , Quimiotaxis/efectos de los fármacos , Cricetinae , Cricetulus , Diseño de Fármacos , Humanos , Hidrazinas/química , Hidrazinas/farmacología , Interleucina-8/metabolismo , Microsomas Hepáticos/metabolismo , Ratas , Receptores de Interleucina-8B/metabolismo , Relación Estructura-ActividadRESUMEN
Farnesyl:protein transferase (FPTase) inhibitors were developed as anti-Ras drugs, but they fail to inhibit Ki-Ras activity because Ki-Ras can be modified by geranylgeranyl:protein transferase type-I (GGPTase-I). L-778,123, an inhibitor of FPTase and GGPTase-I, was developed in part because it can completely inhibit Ki-Ras prenylation. To support the clinical development of L-778,123, we developed pharmacodynamic assays using peripheral blood mononuclear cells (PBMCs) to measure the inhibition of prenylation of HDJ2 and Rap1A, proteins that are FPTase- and GGPTase-I substrates, respectively. We validated these assays in animal models and show that inhibition of HDJ2 prenylation in mouse PBMCs correlates with the concentration of FPTase inhibitors in blood. In dogs, continuous infusion of L-778,123 inhibited both HDJ2 and Rap1A prenylation in PBMCs, but we did not detect inhibition of Ki-Ras prenylation. We reported previously results from the first L-778,123 Phase I trial that showed a dose-dependent inhibition of HDJ2 farnesylation in PBMCs. In this report, we present additional analysis of patient samples from this trial and a second Phase I trial of L-778,123, and demonstrate the inhibition of both HDJ2 and Rap1A prenylation in PBMC samples. This study represents the first demonstration of GGPTase-I inhibition in humans. However, no inhibition of Ki-Ras prenylation by L-778,123 was detected in patient samples. These results confirm the pharmacologic profile of L-778,123 in humans as a dual inhibitor of FPTase and GGPTase-I, but indicate that the intended target of the drug, Ki-Ras, was not inhibited.
Asunto(s)
Transferasas Alquil y Aril/antagonistas & inhibidores , Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Imidazoles/farmacología , Animales , Perros , Relación Dosis-Respuesta a Droga , Humanos , Immunoblotting , Leucocitos/efectos de los fármacos , Leucocitos Mononucleares/efectos de los fármacos , Ratones , Modelos Químicos , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Factores de Tiempo , Proteínas de Unión al GTP rap1/metabolismoRESUMEN
A series of novel diaryl ether lactams have been identified as very potent dual inhibitors of protein farnesyltransferase (FTase) and protein geranylgeranyltransferase I (GGTase-I), enzymes involved in the prenylation of Ras. The structure of the complex formed between one of these compounds and FTase has been determined by X-ray crystallography. These compounds are the first reported to inhibit the prenylation of the important oncogene Ki-Ras4B in vivo. Unfortunately, doses sufficient to achieve this endpoint were rapidly lethal.
Asunto(s)
Transferasas Alquil y Aril/antagonistas & inhibidores , Antineoplásicos/síntesis química , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Proteínas Portadoras/metabolismo , Cristalografía por Rayos X , Ensayos de Selección de Medicamentos Antitumorales , Proteínas del Choque Térmico HSP40 , Proteínas de Choque Térmico/metabolismo , Humanos , Ratones , Ratones Desnudos , Modelos Moleculares , Trasplante de Neoplasias , Neoplasias Experimentales/tratamiento farmacológico , Prenilación de Proteína , Relación Estructura-Actividad , Trasplante Heterólogo , Células Tumorales Cultivadas , Proteínas de Unión al GTP rap1/metabolismo , Proteínas ras/metabolismoRESUMEN
A series of macrocyclic 3-aminopyrrolidinone farnesyltransferase inhibitors (FTIs) has been synthesized. Compared with previously described linear 3-aminopyrrolidinone FTIs such as compound 1, macrocycles such as 49 combined improved pharmacokinetic properties with a reduced potential for side effects. In dogs, oral bioavailability was good to excellent, and increases in plasma half-life were due to attenuated clearance. It was observed that in vivo clearance correlated with the flexibility of the molecules and this concept proved useful in the design of FTIs that exhibited low clearance, such as FTI 78. X-ray crystal structures of compounds 49 and 66 complexed with farnesyltransferase (FTase)-farnesyl diphosphate (FPP) were determined, and they provide details of the key interactions in such ternary complexes. Optimization of this 3-aminopyrrolidinone series of compounds led to significant increases in potency, providing 83 and 85, the most potent inhibitors of FTase in cells described to date.
Asunto(s)
Transferasas Alquil y Aril/antagonistas & inhibidores , Hidrocarburo de Aril Hidroxilasas , Proteínas de Transporte de Catión , Proteínas de Unión al ADN , Inhibidores Enzimáticos/síntesis química , Naftalenos/síntesis química , Canales de Potasio con Entrada de Voltaje , Pirrolidinas/síntesis química , Transactivadores , Animales , Línea Celular , Cromatografía Liquida , Cristalografía por Rayos X , Citocromo P-450 CYP3A , Inhibidores Enzimáticos del Citocromo P-450 , Perros , Canal de Potasio ERG1 , Electrocardiografía , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Canales de Potasio Éter-A-Go-Go , Farnesiltransferasa , Humanos , Técnicas In Vitro , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Modelos Moleculares , Estructura Molecular , Naftalenos/química , Naftalenos/farmacocinética , Oxidorreductasas N-Desmetilantes/antagonistas & inhibidores , Canales de Potasio/metabolismo , Unión Proteica , Pirrolidinas/química , Pirrolidinas/farmacocinética , Estereoisomerismo , Relación Estructura-Actividad , Regulador Transcripcional ERGRESUMEN
Farnesyl:protein transferase (FPTase) catalyzes the covalent addition of the isoprenyl moiety of farnesylpyrophosphate to the C-terminus of the Ras oncoprotein and other cellular proteins. Inhibitors of FPTase (FTIs) have been developed as potential anticancer agents, and several compounds have been evaluated in clinical trials. To facilitate the identification of cell-active FTIs with high potency, the authors developed a method that uses a radiolabeled FTI that serves as a ligand in competitive displacement assays. Using high-affinity [(3)H]-labeled or [(125)I]-labeled FTI radioligands, they show that specific binding to FPTase can be detected in intact cells. Binding of these labeled FTI radioligands can be competed with a variety of structurally diverse FTIs, and the authors show that inhibition of FTI radioligand binding correlates well with inhibition of FPTase substrate prenylation in cells. This method provides a rapid and quantitative means of assessing FTI potency in cells and is useful for guiding the discovery of potent, novel inhibitors of FPTase. Similar methods could be employed in the optimization of inhibitors for other intracellular drug targets.
Asunto(s)
Transferasas Alquil y Aril/antagonistas & inhibidores , Antineoplásicos/farmacología , Ensayos de Selección de Medicamentos Antitumorales/métodos , Inhibidores Enzimáticos/farmacología , Ensayo de Unión Radioligante , Transferasas Alquil y Aril/metabolismo , Animales , Antineoplásicos/química , Antineoplásicos/metabolismo , Línea Celular Transformada , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Farnesiltransferasa , Humanos , Células KB , Ratas , Sensibilidad y EspecificidadRESUMEN
Inhibition of kinesin spindle protein (KSP) is a novel mechanism for treatment of cancer with the potential to overcome limitations associated with currently employed cytotoxic agents. Herein, we describe a C2-hydroxymethyl dihydropyrrole KSP inhibitor ( 11) that circumvents hERG channel binding and poor in vivo potency, issues that limited earlier compounds from our program. However, introduction of the C2-hydroxymethyl group caused 11 to be a substrate for cellular efflux by P-glycoprotein (Pgp). Utilizing knowledge garnered from previous KSP inhibitors, we found that beta-fluorination modulated the p K a of the piperidine nitrogen and reduced Pgp efflux, but the resulting compound ( 14) generated a toxic metabolite in vivo. Incorporation of fluorine in a strategic, metabolically benign position by synthesis of an N-methyl-3-fluoro-4-(aminomethyl)piperidine urea led to compound 30 that has an optimal in vitro and metabolic profile. Compound 30 (MK-0731) was recently studied in a phase I clinical trial in patients with taxane-refractory solid tumors.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Cinesinas/antagonistas & inhibidores , Neoplasias/enzimología , Piperidinas/farmacología , Pirroles/farmacología , Taxoides/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antineoplásicos/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Humanos , Neoplasias/tratamiento farmacológico , Taxoides/uso terapéuticoRESUMEN
3,5-diaryl-4,5-dihydropyrazoles were discovered to be potent KSP inhibitors with excellent in vivo potency. These enzyme inhibitors possess desirable physical properties that can be readily modified by incorporation of a weakly basic amine. Careful adjustment of amine basicity was essential for preserving cellular potency in a multidrug resistant cell line while maintaining good aqueous solubility.
Asunto(s)
Amidas/síntesis química , Amidas/farmacología , Antimitóticos/síntesis química , Antimitóticos/farmacología , Cinesinas/antagonistas & inhibidores , Mitosis/efectos de los fármacos , Pirazoles/síntesis química , Pirazoles/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Línea Celular Tumoral , Fenómenos Químicos , Química Física , Diseño de Fármacos , Resistencia a Antineoplásicos , Genes MDR/efectos de los fármacos , Humanos , Indicadores y Reactivos , Solubilidad , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Inspired by previous efforts in the pyrazolobenzoxazine class of KSP inhibitors, the design and synthesis of 1,4-diaryl-4,5-dihydropyrazole inhibitors of KSP are described. Crystallographic evidence of binding mode and in vivo potency data is also highlighted.
Asunto(s)
Diseño de Fármacos , Hidrógeno/química , Cinesinas/antagonistas & inhibidores , Cinesinas/metabolismo , Mitosis/efectos de los fármacos , Pirazoles/química , Pirazoles/farmacología , Sitios de Unión , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Estructura Molecular , Pirazoles/síntesis química , Relación Estructura-ActividadRESUMEN
Installation of a C2-aminopropyl side chain to the 2,4-diaryl-2,5-dihydropyrrole series of kinesin spindle protein (KSP) inhibitors results in potent, water soluble compounds, but the aminopropyl group induces susceptibility to cellular efflux by P-glycoprotein (Pgp). We show that by carefully modulating the basicity of the amino group by beta-fluorination, this series of inhibitors maintains potency against KSP and has greatly improved efficacy in a Pgp-overexpressing cell line. The discovery that cellular efflux by Pgp can be overcome by carefully modulating the basicity of an amine may be of general use to medicinal chemists attempting to transform leading compounds into cancer cell- or CNS-penetrant drugs.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Flúor/metabolismo , Cinesinas/antagonistas & inhibidores , Propilaminas/farmacología , Pirroles/farmacología , Transporte Biológico , Citoesqueleto , Concentración de Iones de Hidrógeno , Cinesinas/metabolismo , Solubilidad , AguaRESUMEN
The development of 2,5-dihydro-4H-pyrazolo[4,3-c]quinolin-4-ones as inhibitors of Chk1 kinase is described. Introduction of a fused ring at the C7/C8 positions of the pyrazoloquinolinone provided an increase in potency while guidance from overlapping inhibitor bound Chk1 X-ray crystal structures contributed to the discovery of a potent and solubilizing propyl amine moiety in compound 52 (Chk1 IC(50)=3.1 nM).
Asunto(s)
Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/efectos de los fármacos , Quinolonas/farmacología , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Cristalografía por Rayos X , Modelos Moleculares , Inhibidores de Proteínas Quinasas/química , Quinolonas/química , Relación Estructura-ActividadRESUMEN
Observations from two structurally related series of KSP inhibitors led to the proposal and discovery of dihydropyrazolobenzoxazines that possess ideal properties for cancer drug development. The synthesis and characterization of this class of inhibitors along with relevant pharmacokinetic and in vivo data are presented. The synthesis is highlighted by a key [3+2] cycloaddition to form the pyrazolobenzoxazine core followed by diastereospecific installation of a quaternary center.
Asunto(s)
Benzoxazinas/química , Benzoxazinas/farmacología , Diseño de Fármacos , Cinesinas/antagonistas & inhibidores , Cinesinas/metabolismo , Mitosis/efectos de los fármacos , Pirazoles/química , Animales , Benzoxazinas/síntesis química , Benzoxazinas/farmacocinética , Línea Celular , Perros , Humanos , Hidrógeno/química , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Molecular modeling in combination with X-ray crystallographic information was employed to identify a region of the kinesin spindle protein (KSP) binding site not fully utilized by our first generation inhibitors. We discovered that by appending a propylamine substituent at the C5 carbon of a dihydropyrazole core, we could effectively fill this unoccupied region of space and engage in a hydrogen-bonding interaction with the enzyme backbone. This change led to a second generation compound with increased potency, a 400-fold enhancement in aqueous solubility at pH 4, and improved dog pharmacokinetics relative to the first generation compound.
Asunto(s)
Diseño de Fármacos , Cinesinas/antagonistas & inhibidores , Pirazoles/química , Pirazoles/farmacología , Alquilación , Sitio Alostérico , Aminación , Animales , Cristalografía por Rayos X , Perros , Hidroxilación , Cinesinas/química , Cinesinas/metabolismo , Mitosis , Modelos Moleculares , Estructura Molecular , Pirazoles/síntesis química , Pirazoles/farmacocinética , Solubilidad , Relación Estructura-Actividad , AguaRESUMEN
The evolution of 2,4-diaryl-2,5-dihydropyrroles as inhibitors of KSP is described. Introduction of basic amide and urea moieties to the dihydropyrrole nucleus enhanced potency and aqueous solubility, simultaneously, and provided compounds that caused mitotic arrest of A2780 human ovarian carcinoma cells with EC(50)s<10nM. Ancillary hERG activity was evaluated for this series of inhibitors.
Asunto(s)
Cinesinas/antagonistas & inhibidores , Pirroles/química , Pirroles/farmacología , Línea Celular Tumoral , Femenino , Humanos , Modelos Moleculares , Neoplasias Ováricas/patología , Pirroles/síntesis química , Huso Acromático/químicaRESUMEN
2,4-Diaryl-2,5-dihydropyrroles have been discovered to be novel, potent and water-soluble inhibitors of KSP, an emerging therapeutic target for the treatment of cancer. A potential concern for these basic KSP inhibitors (1 and 2) was hERG binding that can be minimized by incorporation of a potency-enhancing C2 phenol combined with neutral N1 side chains. Aqueous solubility was restored to these, and other, non-basic inhibitors, through a phosphate prodrug strategy.
Asunto(s)
Canales de Potasio Éter-A-Go-Go/metabolismo , Cinesinas/antagonistas & inhibidores , Profármacos , Pirroles/síntesis química , Pirroles/farmacología , Animales , Área Bajo la Curva , Perros , Unión Proteica , Pirroles/metabolismo , Pirroles/farmacocinética , Ratas , Solubilidad , Huso Acromático/química , AguaRESUMEN
The development of 3-(indol-2-yl)indazoles as inhibitors of Chek1 kinase is described. Introduction of amides and heteroaryl groups at the C6 position of the indazole ring system provided sufficient Chek1 potency and selectivity over Cdk7 to permit escape from DNA damage-induced arrest in a cellular assay. Enzyme potency against Chek1 was optimized by the incorporation of a hydroxymethyl triazole moiety in compound 21 (Chek1 IC(50)=0.30nM) that was shown by X-ray crystallography to displace one of three highly conserved water molecules in the HI region of the ATP-binding cleft.
Asunto(s)
Indazoles/química , Indazoles/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Cristalografía por Rayos X , Humanos , Indazoles/metabolismo , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/metabolismo , Relación Estructura-ActividadRESUMEN
Compound 1 has been shown to be a dual prenylation inhibitor with FPTase (IC50=2 nM) and GGPTase-I (IC50=95 nM). Analogues of 1, which replaced the cyanophenyl group with various biaryls, led to the discovery of highly potent dual FPTase/GGPTase-I inhibitors. 4-trifluoromethylphenyl, trifluoropentynyl, and trifluoropentyl were identified as good p-cyano replacements.