RESUMO
The nonclassical extracellular signal-related kinase 5 (ERK5) mitogen-activated protein kinase pathway has been implicated in increased cellular proliferation, migration, survival, and angiogenesis; hence, ERK5 inhibition may be an attractive approach for cancer treatment. However, the development of selective ERK5 inhibitors has been challenging. Previously, we described the development of a pyrrole carboxamide high-throughput screening hit into a selective, submicromolar inhibitor of ERK5 kinase activity. Improvement in the ERK5 potency was necessary for the identification of a tool ERK5 inhibitor for target validation studies. Herein, we describe the optimization of this series to identify nanomolar pyrrole carboxamide inhibitors of ERK5 incorporating a basic center, which suffered from poor oral bioavailability. Parallel optimization of potency and in vitro pharmacokinetic parameters led to the identification of a nonbasic pyrazole analogue with an optimal balance of ERK5 inhibition and oral exposure.
Assuntos
Proteína Quinase 7 Ativada por Mitógeno , Pirróis , Proliferação de Células , Pirróis/farmacologiaRESUMO
Inhibition of murine double minute 2 (MDM2)-p53 protein-protein interaction with small molecules has been shown to reactivate p53 and inhibit tumor growth. Here, we describe rational, structure-guided, design of novel isoindolinone-based MDM2 inhibitors. MDM2 X-ray crystallography, quantum mechanics ligand-based design, and metabolite identification all contributed toward the discovery of potent in vitro and in vivo inhibitors of the MDM2-p53 interaction with representative compounds inducing cytostasis in an SJSA-1 osteosarcoma xenograft model following once-daily oral administration.
Assuntos
Antineoplásicos/farmacologia , Isoindóis/farmacologia , Osteossarcoma/tratamento farmacológico , Multimerização Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Neoplasias Ósseas/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Estabilidade de Medicamentos , Feminino , Humanos , Isoindóis/síntese química , Isoindóis/metabolismo , Macaca fascicularis , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Ligação Proteica , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Renewed interest in covalent inhibitors of enzymes implicated in disease states has afforded several agents targeted at protein kinases of relevance to cancers. We now report the design, synthesis and biological evaluation of 6-ethynylpurines that act as covalent inhibitors of Nek2 by capturing a cysteine residue (Cys22) close to the catalytic domain of this protein kinase. Examination of the crystal structure of the non-covalent inhibitor 3-((6-cyclohexylmethoxy-7H-purin-2-yl)amino)benzamide in complex with Nek2 indicated that replacing the alkoxy with an ethynyl group places the terminus of the alkyne close to Cys22 and in a position compatible with the stereoelectronic requirements of a Michael addition. A series of 6-ethynylpurines was prepared and a structure activity relationship (SAR) established for inhibition of Nek2. 6-Ethynyl-N-phenyl-7H-purin-2-amine [IC50 0.15 µM (Nek2)] and 4-((6-ethynyl-7H-purin-2-yl)amino)benzenesulfonamide (IC50 0.14 µM) were selected for determination of the mode of inhibition of Nek2, which was shown to be time-dependent, not reversed by addition of ATP and negated by site directed mutagenesis of Cys22 to alanine. Replacement of the ethynyl group by ethyl or cyano abrogated activity. Variation of substituents on the N-phenyl moiety for 6-ethynylpurines gave further SAR data for Nek2 inhibition. The data showed little correlation of activity with the nature of the substituent, indicating that after sufficient initial competitive binding to Nek2 subsequent covalent modification of Cys22 occurs in all cases. A typical activity profile was that for 2-(3-((6-ethynyl-9H-purin-2-yl)amino)phenyl)acetamide [IC50 0.06 µM (Nek2); GI50 (SKBR3) 2.2 µM] which exhibited >5-10-fold selectivity for Nek2 over other kinases; it also showed > 50% growth inhibition at 10 µM concentration against selected breast and leukaemia cell lines. X-ray crystallographic analysis confirmed that binding of the compound to the Nek2 ATP-binding site resulted in covalent modification of Cys22. Further studies confirmed that 2-(3-((6-ethynyl-9H-purin-2-yl)amino)phenyl)acetamide has the attributes of a drug-like compound with good aqueous solubility, no inhibition of hERG at 25 µM and a good stability profile in human liver microsomes. It is concluded that 6-ethynylpurines are promising agents for cancer treatment by virtue of their selective inhibition of Nek2.
RESUMO
Potentiating radiotherapy and chemotherapy by inhibiting DNA damage repair is proposed as a therapeutic strategy to improve outcomes for patients with solid tumors. However, this approach risks enhancing normal tissue toxicity as much as tumor toxicity, thereby limiting its translational impact. Using NU5455, a newly identified highly selective oral inhibitor of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity, we found that it was indeed possible to preferentially augment the effect of targeted radiotherapy on human orthotopic lung tumors without influencing acute DNA damage or a late radiation-induced toxicity (fibrosis) to normal mouse lung. Furthermore, while NU5455 administration increased both the efficacy and the toxicity of a parenterally administered topoisomerase inhibitor, it enhanced the activity of doxorubicin released locally in liver tumor xenografts without inducing any adverse effect. This strategy is particularly relevant to hepatocellular cancer, which is treated clinically with localized drug-eluting beads and for which DNA-PKcs activity is reported to confer resistance to treatment. We conclude that transient pharmacological inhibition of DNA-PKcs activity is effective and tolerable when combined with localized DNA-damaging therapies and thus has promising clinical potential.
Assuntos
Carcinoma Hepatocelular , Proteína Quinase Ativada por DNA/antagonistas & inibidores , Neoplasias Hepáticas Experimentais , Proteínas de Neoplasias/antagonistas & inibidores , Inibidores de Proteínas Quinases , Animais , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/patologia , Proteína Quinase Ativada por DNA/metabolismo , Doxorrubicina/farmacologia , Humanos , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Neoplasias Hepáticas Experimentais/enzimologia , Neoplasias Hepáticas Experimentais/patologia , Células MCF-7 , Camundongos , Proteínas de Neoplasias/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Extracellular regulated kinase 5 (ERK5) signalling has been implicated in driving a number of cellular phenotypes including endothelial cell angiogenesis and tumour cell motility. Novel ERK5 inhibitors were identified using high throughput screening, with a series of pyrrole-2-carboxamides substituted at the 4-position with an aroyl group being found to exhibit IC50 values in the micromolar range, but having no selectivity against p38α MAP kinase. Truncation of the N-substituent marginally enhanced potency (â¼3-fold) against ERK5, but importantly attenuated inhibition of p38α. Systematic variation of the substituents on the aroyl group led to the selective inhibitor 4-(2-bromo-6-fluorobenzoyl)-N-(pyridin-3-yl)-1H-pyrrole-2-carboxamide (IC50 0.82⯵M for ERK5; IC50â¯>â¯120⯵M for p38α). The crystal structure (PDB 5O7I) of this compound in complex with ERK5 has been solved. This compound was orally bioavailable and inhibited bFGF-driven Matrigel plug angiogenesis and tumour xenograft growth. The selective ERK5 inhibitor described herein provides a lead for further development into a tool compound for more extensive studies seeking to examine the role of ERK5 signalling in cancer and other diseases.
Assuntos
Antineoplásicos/farmacologia , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 7 Ativada por Mitógeno/antagonistas & inibidores , Proteínas Nucleares/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Disponibilidade Biológica , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Camundongos , Camundongos Nus , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Proteínas Nucleares/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismoRESUMO
Nek2 (NIMA-related kinase 2) is a cell cycle-dependent serine/threonine protein kinase that regulates centrosome separation at the onset of mitosis. Overexpression of Nek2 is common in human cancers and suppression can restrict tumor cell growth and promote apoptosis. Nek2 inhibition with small molecules, therefore, offers the prospect of a new therapy for cancer. To achieve this goal, a better understanding of the requirements for selective-inhibition of Nek2 is required. 6-Alkoxypurines were identified as ATP-competitive inhibitors of Nek2 and CDK2. Comparison with CDK2-inhibitor structures indicated that judicious modification of the 6-alkoxy and 2-arylamino substituents could achieve discrimination between Nek2 and CDK2. In this study, a library of 6-cyclohexylmethoxy-2-arylaminopurines bearing carboxamide, sulfonamide and urea substituents on the 2-arylamino ring was synthesized. Few of these compounds were selective for Nek2 over CDK2, with the best result being obtained for 3-((6-(cyclohexylmethoxy)-9H-purin-2-yl)amino)-N,N-dimethylbenzamide (CDK2 IC50 = 7.0 µM; Nek2 IC50 = 0.62 µM) with >10-fold selectivity. Deletion of the 6-substituent abrogated activity against both Nek2 and CDK2. Nine compounds containing an (E)-dialkylaminovinyl substituent at C-6, all showed selectivity for Nek2, e.g. (E)-6-(2-(azepan-1-yl)vinyl)-N-phenyl-9H-purin-2-amine (CDK2 IC50 = 2.70 µM; Nek2 IC50 = 0.27 µM). Structural biology of selected compounds enabled a partial rationalization of the observed structure activity relationships and mechanism of Nek2 activation. This showed that carboxamide 11 is the first reported inhibitor of Nek2 in the DFG-in conformation.
Assuntos
Antineoplásicos/farmacologia , Desenho de Fármacos , Quinases Relacionadas a NIMA/antagonistas & inibidores , Antineoplásicos/química , Linhagem Celular Tumoral , Humanos , Espectroscopia de Ressonância Magnética , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Examining essential biochemical pathways in Plasmodium falciparum presents serious challenges, as standard molecular techniques such as siRNA cannot be employed in this organism, and generating gene knock-outs of essential proteins requires specialized conditional approaches. In the study of protein kinases, pharmacological inhibition presents a feasible alternative option. However, as in mammalian systems, inhibitors often lack the desired selectivity. Described here is a chemical genetic approach to selectively inhibit Pfnek-2 in P. falciparum, a member of the NIMA-related kinase family that is essential for completion of the sexual development of the parasite. RESULTS: Introduction of a valine to cysteine mutation at position 24 in the glycine rich loop of Pfnek-2 does not affect kinase activity but confers sensitivity to the protein kinase inhibitor 4-(6-ethynyl-9H-purin-2-ylamino) benzene sulfonamide (NCL-00016066). Using a combination of in vitro kinase assays and mass spectrometry, (including phosphoproteomics) the study shows that this compound acts as an irreversible inhibitor to the mutant Pfnek2 likely through a covalent link with the introduced cysteine residue. In particular, this was shown by analysis of total protein mass using mass spectrometry which showed a shift in molecular weight of the mutant kinase in the presence of the inhibitor to be precisely equivalent to the molecular weight of NCL-00016066. A similar molecular weight shift was not observed in the wild type kinase. Importantly, this inhibitor has little activity towards the wild type Pfnek-2 and, therefore, has all the properties of an effective chemical genetic tool that could be employed to determine the cellular targets for Pfnek-2. CONCLUSIONS: Allelic replacement of wild-type Pfnek-2 with the mutated kinase will allow for targeted inhibition of Pfnek-2 with NCL-00016066 and hence pave the way for comparative studies aimed at understanding the biological role and transmission-blocking potential of Pfnek-2.
Assuntos
Inibidores Enzimáticos/metabolismo , Proteínas Mutantes/metabolismo , Quinases Relacionadas a NIMA/metabolismo , Plasmodium falciparum/enzimologia , Purinas/metabolismo , Sulfonamidas/metabolismo , Espectrometria de Massas , Proteínas Mutantes/genética , Quinases Relacionadas a NIMA/genéticaRESUMO
BACKGROUND: The antimalarial drug Pyrimethamine has been suggested to exert an antitumor activity by inducing apoptotic cell death in cancer cells, including metastatic melanoma cells. However, the dose of Pyrimethamine to be considered as an anticancer agent appears to be significantly higher than the maximum dose used as an antiprotozoal drug. METHODS: Hence, a series of Pyrimethamine analogs has been synthesized and screened for their apoptosis induction in two cultured metastatic melanoma cell lines. One of these analogs, the Methylbenzoprim, was further analyzed to evaluate cell-cycle and the mechanisms of cell death. The effects of Methylbenzoprim were also analyzed in a severe combined immunodeficiency (SCID)-mouse xenotransplantation model. RESULTS: Low dose of Methylbenzoprim was capable of inducing cytotoxic activity and a potent growth-inhibitory effect by arresting cell cycle in S-phase in melanoma cells. Methylbenzoprim was also detected as powerful antineoplastic agents in SCID-mouse although used at very low dose and as a single agent. CONCLUSIONS: Our screening approach led to the identification of a "low cost" newly synthesized drug (methylbenzoprim), which is able to act as an antineoplastic agent in vitro and in vivo, inhibiting melanoma tumor growth at very low concentrations.
Assuntos
Antineoplásicos/administração & dosagem , Melanoma/tratamento farmacológico , Pirimetamina/análogos & derivados , Pirimidinas/administração & dosagem , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Melanoma/metabolismo , Camundongos , Camundongos SCID , Metástase Neoplásica , Pirimidinas/síntese química , Pirimidinas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: The phosphatidylinositol-3-kinase/mammalian target of rapamycin (PI3K/mTOR) pathway is commonly deregulated in human cancer, hence many PI3K and mTOR inhibitors have been developed and have now reached clinical trials. Similarly, CDKs have been investigated as cancer drug targets. METHODS: We have synthesised and characterised a series of 6-aminopyrimidines identified from a kinase screen that inhibit PI3K and/or mTOR and/or CDK2. Kinase inhibition, tumour cell growth, cell cycle distribution, cytotoxicity and signalling experiments were undertaken in HCT116 and HT29 colorectal cancer cell lines, and in vivo HT29 efficacy studies. RESULTS: 2,6-Diaminopyrimidines with an O(4)-cyclohexylmethyl substituent and a C-5-nitroso or cyano group (1,2,5) induced cell cycle phase alterations and were growth inhibitory (GI50<20 µM). Compound 1, but not 2 or 5, potently inhibits CDK2 (IC50=0.1 nM) as well as PI3K, and was cytotoxic at growth inhibitory concentrations. Consistent with kinase inhibition data, compound 1 reduced phospho-Rb and phospho-rS6 at GI50 concentrations. Combination of NU6102 (CDK2 inhibitor) and pictilisib (GDC-0941; pan-PI3K inhibitor) resulted in synergistic growth inhibition, and enhanced cytotoxicity in HT29 cells in vitro and HT29 tumour growth inhibition in vivo. CONCLUSIONS: These studies identified a novel series of mixed CDK2/PI3K inhibitors and demonstrate that dual targeting of CDK2 and PI3K can result in enhanced antitumour activity.
Assuntos
Adenocarcinoma/patologia , Neoplasias Colorretais/patologia , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Terapia de Alvo Molecular , Proteínas de Neoplasias/antagonistas & inibidores , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Adenocarcinoma/enzimologia , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias Colorretais/enzimologia , Quinase 2 Dependente de Ciclina/fisiologia , Humanos , Camundongos , Camundongos Nus , Proteínas de Neoplasias/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/síntese química , Pirimidinas/farmacologia , Distribuição Aleatória , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Ensaio Tumoral de Célula-Tronco , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The extracellular-related kinase 5 (ERK5) is a promising target for cancer therapy. A high-throughput screen was developed for ERK5, based on the IMAP FP progressive binding system, and used to identify hits from a library of 57â¯617 compounds. Four distinct chemical series were evident within the screening hits. Resynthesis and reassay of the hits demonstrated that one series did not return active compounds, whereas three series returned active hits. Structure-activity studies demonstrated that the 4-benzoylpyrrole-2-carboxamide pharmacophore had excellent potential for further development. The minimum kinase binding pharmacophore was identified, and key examples demonstrated good selectivity for ERK5 over p38α kinase.
Assuntos
Amidas/química , Proteína Quinase 7 Ativada por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Pirróis/química , Amidas/síntese química , Antineoplásicos/síntese química , Antineoplásicos/química , Ensaios de Triagem em Larga Escala , Humanos , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Estrutura Molecular , Neoplasias/tratamento farmacológico , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Pirróis/síntese química , Relação Estrutura-AtividadeRESUMO
Non-genotoxic reactivation of the p53 pathway by MDM2-p53 binding antagonists is an attractive treatment strategy for wild-type TP53 cancers. To determine how resistance to MDM2/p53 binding antagonists might develop, SJSA-1 and NGP cells were exposed to growth inhibitory concentrations of chemically distinct MDM2 inhibitors, Nutlin-3 and MI-63, and clonal resistant cell lines generated. The p53 mediated responses of parental and resistant cell lines were compared. In contrast to the parental cell lines, p53 activation by Nutlin-3, MI-63 or ionizing radiation was not observed in either the SJSA-1 or the NGP derived cell lines. An identical TP53 mutation was subsequently identified in both of the SJSA-1 resistant lines, whilst one out of three identified mutations was common to both NGP derived lines. Mutation specific PCR revealed these mutations were present in parental SJSA-1 and NGP cell populations at a low frequency. Despite cross-resistance to a broad panel of MDM2/p53 binding antagonists, these MDM2-amplified and TP53 mutant cell lines remained sensitive to ionizing radiation (IR). These results indicate that MDM2/p53 binding antagonists will select for p53 mutations present in tumours at a low frequency at diagnosis, leading to resistance, but such tumours may nevertheless remain responsive to alternative therapies, including IR.
Assuntos
Resistencia a Medicamentos Antineoplásicos/fisiologia , Resistencia a Medicamentos Antineoplásicos/efeitos da radiação , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteína Supressora de Tumor p53/genética , Linhagem Celular Tumoral , Humanos , Mutação , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteína Supressora de Tumor p53/antagonistas & inibidoresRESUMO
Inhibitors of sulfatase-2 are putative anticancer agents, but the discovery of potent small molecules targeting this enzyme has proved challenging. Based on molecular modelling, two series of sulfatase-2 inhibitors have been developed with biphenyl and biphenyl ether scaffolds judiciously substituted with sulfamate, carboxylate and other polar groups (e.g. amino). Inhibition of aryl sulfatase A and B was also determined. The biphenyl ether derivatives were less selective for sulfatase-2 over aryl sulfatase B than the biphenyl series. All biphenyl ether derivatives inhibited aryl sulfatase A, whereas only amino derivatives inhibited aryl sulfatase B significantly. In the biphenyl series few derivatives exhibited activity against aryl sulfatase B. The trichloroethylsulfamate group was identified as a new pharmacophore enabling potent inhibition of all of the sulfatases studied.
RESUMO
Irreversible inhibitors that modify cysteine or lysine residues within a protein kinase ATP binding site offer, through their distinctive mode of action, an alternative to ATP-competitive agents. 4-((6-(Cyclohexylmethoxy)-9H-purin-2-yl)amino)benzenesulfonamide (NU6102) is a potent and selective ATP-competitive inhibitor of CDK2 in which the sulfonamide moiety is positioned close to a pair of lysine residues. Guided by the CDK2/NU6102 structure, we designed 6-(cyclohexylmethoxy)-N-(4-(vinylsulfonyl)phenyl)-9H-purin-2-amine (NU6300), which binds covalently to CDK2 as shown by a co-complex crystal structure. Acute incubation with NU6300 produced a durable inhibition of Rb phosphorylation in SKUT-1B cells, consistent with it acting as an irreversible CDK2 inhibitor. NU6300 is the first covalent CDK2 inhibitor to be described, and illustrates the potential of vinyl sulfones for the design of more potent and selective compounds.
Assuntos
Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Purinas/química , Purinas/farmacologia , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Ligação Competitiva , Cristalografia por Raios X , Quinase 2 Dependente de Ciclina/química , Quinase 2 Dependente de Ciclina/metabolismo , Desenho de Fármacos , Humanos , Modelos Moleculares , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Purinas/síntese química , Relação Estrutura-Atividade , Sulfonas/químicaRESUMO
Two libraries of substituted benzimidazoles were designed using a 'scaffold-hopping' approach based on reported MDM2-p53 inhibitors. Substituents were chosen following library enumeration and docking into an MDM2 X-ray structure. Benzimidazole libraries were prepared using an efficient solution-phase approach and screened for inhibition of the MDM2-p53 and MDMX-p53 protein-protein interactions. Key examples showed inhibitory activity against both targets.
Assuntos
Benzimidazóis/farmacologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteína Supressora de Tumor p53/antagonistas & inibidores , Benzimidazóis/química , Proteínas de Ciclo Celular , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismoRESUMO
Recent studies have shown that irreversible inhibition of Nek2 kinase [(Never in mitosis gene a)-related kinase 2], overexpression of which is observed in several cancers, can be achieved using Michael acceptors containing an ethynyl group, which target the enzyme's cysteine 22 residue lying near the catalytic site. The model studies described herein demonstrate an analogous capture of the ethynyl moiety in a series of ethynyl-heterocycles (e.g. 6-ethynyl-N-phenyl-9H-purin-2-amine) by N-acetylcysteine methyl ester in the presence of 1,4-diazabicyclo[2.2.2]octane in either dimethyl sulfoxide or N,N-dimethylformamide. Kinetic studies showed a 50-fold range in reactivity with 7-ethynyl-N-phenyl-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-amine being the most reactive compound, whereas 4-ethynyl-N-phenyl-7H-pyrrolo[2,3-d]pyrimidin-2-amine was the least reactive. Studies of the isomeric compounds, 2-(3-((6-ethynyl-7-methyl-7H-purin-2-yl)amino)phenyl)acetamide and 2-(3-((6-ethynyl-9-methyl-9H-purin-2-yl)amino)phenyl)acetamide, revealed the N(7)-methyl isomer to be 5-fold more reactive than the 9-methyl isomer, which is ascribed to a buttressing effect in the N(7)-methyl compound. Comparison of the crystal structures of these isomers showed that the ethynyl group is significantly displaced away from the methyl group exclusively in the N(7)-methyl isomer with an sp(2) bond angle of 124°, whereas the corresponding angle in the N(9)-methyl isomer was the expected 120°. The results of this study indicate heterocyclic scaffolds that are likely to be more promising for inhibition of Nek2 and other kinases containing a reactive cysteine.
Assuntos
Compostos Heterocíclicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Purinas/farmacologia , Compostos de Sulfidrila/química , Cristalografia por Raios X , Compostos Heterocíclicos/química , Humanos , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , Quinases Relacionadas a NIMA , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Purinas/químicaRESUMO
The ribosomal P70 S6 kinases play a crucial role in PI3K/mTOR regulated signalling pathways and are therefore potential targets for the treatment of a variety of diseases including diabetes and cancer. In this study we describe the identification of three series of chemically distinct S6K1 inhibitors. In addition, we report a novel PKA-S6K1 chimeric protein with five mutations in or near its ATP-binding site, which was used to determine the binding mode of two of the three inhibitor series, and provided a robust system to aid the optimisation of the oxadiazole-substituted benzimidazole inhibitor series. We show that the resulting oxadiazole-substituted aza-benzimidazole is a potent and ligand efficient S6 kinase inhibitor, which blocks the phosphorylation of RPS6 at Ser235/236 in TSC negative HCV29 human bladder cancer cells by inhibiting S6 kinase activity and thus provides a useful tool compound to investigate the function of S6 kinases.
Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Linhagem Celular Tumoral , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Desenho de Fármacos , Ensaios de Triagem em Larga Escala/métodos , Humanos , Imidazóis/química , Imidazóis/farmacologia , Modelos Moleculares , Fosforilação , Inibidores de Proteínas Quinases/química , Proteínas Recombinantes de Fusão/química , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/enzimologiaRESUMO
Analogues of (dibenzo[b,d]thiophen-4-yl)-2-morpholino-4H-chromen-4-one (NU7441), a potent inhibitor of DNA-dependent protein kinase (DNA-PK; IC50 = 42 ± 2 nM), have been synthesized in which water-solubilizing groups [NHCO(CH2)nNR¹R², where n = 1 or 2 and the moiety R¹R²N was derived from a library of primary and secondary amines, e.g., morpholine] were placed at the 1-position. Several of the newly synthesized compounds exhibited high potency against DNA-PK and potentiated the cytotoxicity of ionizing radiation (IR) in vitro 10-fold or more (e.g., 2-(4-ethylpiperazin-1-yl)-N-(4-(2-morpholino-4-oxo-4H-chromen-8-yl)dibenzo[b,d]thio-phen-1-yl)acetamide, 39; DNA-PK IC50 = 5.0 ± 1 nM, IR dose modification ratio = 13). Furthermore, 39 was shown to potentiate not only IR in vitro but also DNA-inducing cytotoxic anticancer agents, both in vitro and in vivo. Counter-screening against other members of the phosphatidylinositol 3-kinase (PI-3K) related kinase (PIKK) family unexpectedly revealed that some of the compounds were potent mixed DNA-PK and PI-3K inhibitors.
Assuntos
Proteína Quinase Ativada por DNA/antagonistas & inibidores , Morfolinas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Células HeLa , Humanos , Morfolinas/químicaRESUMO
Tip60 (KAT5) is a histone acetyltransferase (HAT enzyme) involved in multiple cellular processes including transcriptional regulation, DNA damage repair and cell signalling. In prostate cancer, aggressive cases over-express Tip60 which functions as an androgen receptor co-activator via direct acetylation of lysine residues within the KLKK motif of the receptor hinge region. The purpose of this study was to identify and characterise a Tip60 acetylase inhibitor. High-throughput screening revealed an isothiazole that inhibited both Tip60 and p300 HAT activity. This substance (initially identified as 4-methyl-5-bromoisothiazole) and other isothiazoles were synthesised and assayed against Tip60. Although an authentic sample of 4-methyl-5-bromoisothiazole was inactive against Tip60, in an in vitro HAT assay, 1,2-bis(isothiazol-5-yl)disulfane (NU9056) was identified as a relatively potent inhibitor (IC(50) 2 µM). Cellular activity was confirmed by analysis of acetylation of histone and non-histone proteins in a prostate cancer cell line model. NU9056 treatment inhibited cellular proliferation in a panel of prostate cancer cell lines (50% growth inhibition, 8-27 µM) and induced apoptosis via activation of caspase 3 and caspase 9 in a concentration- and time-dependent manner. Also, decreased androgen receptor, prostate specific antigen, p53 and p21 protein levels were demonstrated in response to treatment with NU9056. Furthermore, pre-treatment with NU9056 inhibited both ATM phosphorylation and Tip60 stabilization in response to ionising radiation. Based on the activity of NU9056 and the specificity of the compound towards Tip60 relative to other HAT enzymes, these chemical biology studies have identified Tip60 as a potential therapeutic target for the treatment of prostate cancer.
Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Histona Acetiltransferases/antagonistas & inibidores , Tiazóis/farmacologia , Acetilação/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia , Western Blotting , Caspase 3/metabolismo , Caspase 9/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Células HEK293 , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Humanos , Concentração Inibidora 50 , Lisina Acetiltransferase 5 , Masculino , Modelos Químicos , Estrutura Molecular , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Radiação Ionizante , Tiazóis/química , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Modulation of DNA repair pathways in oncology has been an area of intense interest in the last decade, not least as a consequence of the promising clinical activity of poly(ADP-ribose) polymerase (PARP) inhibitors. In this review article, we highlight inhibitors of the phosphatidylinositol 3-kinase related kinase (PIKK) family as of potential interest in the treatment of cancer, both in combination with DNA-damaging therapies and as stand-alone agents.
Assuntos
Reparo do DNA/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , DNA/metabolismo , Proteína Quinase Ativada por DNA/antagonistas & inibidores , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Humanos , Modelos Moleculares , Neoplasias/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/metabolismoRESUMO
DNA double-strand breaks (DSB) are the most cytotoxic lesions induced by topoisomerase II poisons. Nonhomologous end joining (NHEJ) is a major pathway for DSB repair and requires DNA-dependent protein kinase (DNA-PK) activity. DNA-PK catalytic subunit (DNA-PKcs) is structurally similar to PI-3K, which promotes cell survival and proliferation and is upregulated in many cancers. KU-0060648 is a dual inhibitor of DNA-PK and PI-3K in vitro. KU-0060648 was investigated in a panel of human breast and colon cancer cells. The compound inhibited cellular DNA-PK autophosphorylation with IC(50) values of 0.019 µmol/L (MCF7 cells) and 0.17 µmol/L (SW620 cells), and PI-3K-mediated AKT phosphorylation with IC(50) values of 0.039 µmol/L (MCF7 cells) and more than 10 µmol/L (SW620 cells). Five-day exposure to 1 µmol/L KU-0060648 inhibited cell proliferation by more than 95% in MCF7 cells but only by 55% in SW620 cells. In clonogenic survival assays, KU-0060648 increased the cytotoxicity of etoposide and doxorubicin across the panel of DNA-PKcs-proficient cells, but not in DNA-PKcs-deficient cells, thus confirming that enhanced cytotoxicity was due to DNA-PK inhibition. In mice bearing SW620 and MCF7 xenografts, concentrations of KU-0060648 that were sufficient for in vitro growth inhibition and chemosensitization were maintained within the tumor for at least 4 hours at nontoxic doses. KU-0060648 alone delayed the growth of MCF7 xenografts and increased etoposide-induced tumor growth delay in both in SW620 and MCF7 xenografts by up to 4.5-fold, without exacerbating etoposide toxicity to unacceptable levels. The proof-of-principle in vitro and in vivo chemosensitization with KU-0060648 justifies further evaluation of dual DNA-PK and PI-3K inhibitors.