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1.
Br J Cancer ; 115(6): 682-90, 2016 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-27529512

RESUMO

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 Xenoenxerto
2.
J Med Chem ; 65(9): 6513-6540, 2022 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-35468293

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/farmacologia
3.
J Mol Biol ; 433(5): 166795, 2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33422522

RESUMO

The SCFSKP2 ubiquitin ligase relieves G1 checkpoint control of CDK-cyclin complexes by promoting p27KIP1 degradation. We describe reconstitution of stable complexes containing SKP1-SKP2 and CDK1-cyclin B or CDK2-cyclin A/E, mediated by the CDK regulatory subunit CKS1. We further show that a direct interaction between a SKP2 N-terminal motif and cyclin A can stabilize SKP1-SKP2-CDK2-cyclin A complexes in the absence of CKS1. We identify the SKP2 binding site on cyclin A and demonstrate the site is not present in cyclin B or cyclin E. This site is distinct from but overlapping with features that mediate binding of p27KIP1 and other G1 cyclin regulators to cyclin A. We propose that the capacity of SKP2 to engage with CDK2-cyclin A by more than one structural mechanism provides a way to fine tune the degradation of p27KIP1 and distinguishes cyclin A from other G1 cyclins to ensure orderly cell cycle progression.


Assuntos
Ciclina A/química , Quinase 2 Dependente de Ciclina/química , Inibidor de Quinase Dependente de Ciclina p27/química , Pontos de Checagem da Fase G1 do Ciclo Celular , Proteínas Quinases Associadas a Fase S/química , Sítios de Ligação , Quinases relacionadas a CDC2 e CDC28/química , Quinases relacionadas a CDC2 e CDC28/genética , Quinases relacionadas a CDC2 e CDC28/metabolismo , Ciclina A/genética , Ciclina A/metabolismo , Ciclina E/química , Ciclina E/genética , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Quinases Associadas a Fase S/genética , Proteínas Quinases Associadas a Fase S/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais
4.
Org Biomol Chem ; 8(10): 2457-64, 2010 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-20448906

RESUMO

The attenuated S(N)2 reactivity of the 2,2,2-trifluoroethyl group has been exploited for the synthesis of a series of 6-cyclohexylmethoxy-2-arylaminopurines in which a sulfonamide moiety was attached to the aryl ring via a methylene group. These were required as potential inhibitors of serine-threonine kinases of interest for the treatment of cancer. 3-Nitrophenylmethanesulfonyl chloride was converted into the corresponding 2,2,2-trifluoroethoxysulfonyl ester by reaction with 2,2,2-trifluoroethanol in the presence of triethylamine/4-dimethylaminopyridine. Catalytic hydrogenation of the nitro group employing 2,2,2-trifluoroethanol as solvent gave 2,2,2-trifluoroethyl 3-aminophenylmethanesulfonate, which was reacted with 6-cyclohexylmethoxy-2-fluoropurine in 2,2,2-trifluoroethanol/trifluoroacetic acid to afford 2,2,2-trifluoroethyl 3-(6-cyclohexylmethoxy-9H-purin-2-ylamino)phenylmethanesulfonate. 3-(6-Cyclohexylmethoxy-9H-purin-2-ylamino)phenylmethanesulfonamides were synthesised by microwave heating of the trifluoroethoxysulfonate with an amine and 1,8-diazabicycloundec-7-ene in tetrahydrofuran. The mechanism of this process was shown to involve an intermediate sulfene by a deuterium-labelling experiment. 3-(6-Cyclohexylmethoxy-9H-purin-2-ylamino)phenylmethanesulfonamide derivatives were assayed as inhibitors of human cyclin-dependent kinase 2. Previous structure-activity studies demonstrated that relocating the sulfonamide group of O(6)-cyclohexylmethoxy-2-(4'-sulfamoylanilino)purine from the 4- to the 3-position on the 2-arylamino ring resulted in a 40-fold reduction in potency against CDK2. In the present study, no further loss of activity was observed on introducing a methylene group between the sulfonamide and the aryl ring, 3-(6-cyclohexylmethoxy-9H-purin-2-ylamino)phenylmethanesulfonamide proving equipotent with O(6)-cyclohexylmethoxy-2-(3'-sulfamoylanilino)purine (IC(50) = 0.21 microM). N-Alkylation of the sulfonamide reduced CDK-2 inhibitory activity, while a substituted benzyl or 3-phenylpropyl group on the sulfonamide resulted in a loss of potency compared with 3-(6-cyclohexylmethoxy-9H-purin-2-ylamino)phenylmethanesulfonamide. The dimethylaminopropyl derivative, 1-[3-(6-cyclohexylmethoxy-9H-purin-2-ylamino)phenyl]-N-(3-dimethylaminopropyl)methanesulfonamide was only 2-fold less potent than 3-(6-cyclohexylmethoxy-9H-purin-2-ylamino)phenylmethanesulfonamide, suggesting an interaction between the basic dimethylamino group and the kinase. The presence of alicyclic groups on the pendant sulfonamide showed IC(50) values in the 0.5-1.5 microM range. N-(4-tert-Butylphenyl)-1-[3-(6-cyclohexylmethoxy-9H-purin-2-ylamino)phenyl]methanesulfonamide was markedly less active (IC(50) = 34 microM), suggesting a steric effect within the ATP-binding domain.


Assuntos
Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Sulfonamidas/síntese química , Sulfonamidas/farmacologia , Ácidos Sulfônicos/química , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Humanos , Inibidores de Proteínas Quinases/química , Purinas/química , Sulfonamidas/química
5.
Cell Chem Biol ; 26(1): 121-130.e5, 2019 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-30472117

RESUMO

Dysregulation of the cell cycle characterizes many cancer subtypes, providing a rationale for developing cyclin-dependent kinase (CDK) inhibitors. Potent CDK2 inhibitors might target certain cancers in which CCNE1 is amplified. However, current CDK2 inhibitors also inhibit CDK1, generating a toxicity liability. We have used biophysical measurements and X-ray crystallography to investigate the ATP-competitive inhibitor binding properties of cyclin-free and cyclin-bound CDK1 and CDK2. We show that these kinases can readily be distinguished by such inhibitors when cyclin-free, but not when cyclin-bound. The basis for this discrimination is unclear from either inspection or molecular dynamics simulation of ligand-bound CDKs, but is reflected in the contacts made between the kinase N- and C-lobes. We conclude that there is a subtle but profound difference between the conformational energy landscapes of cyclin-free CDK1 and CDK2. The unusual properties of CDK1 might be exploited to differentiate CDK1 from other CDKs in future cancer therapeutic design.


Assuntos
Proteína Quinase CDC2/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Entropia , Inibidores de Proteínas Quinases/farmacologia , Proteína Quinase CDC2/isolamento & purificação , Proteína Quinase CDC2/metabolismo , Quinase 2 Dependente de Ciclina/isolamento & purificação , Quinase 2 Dependente de Ciclina/metabolismo , Humanos , Conformação Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteínas Quinases/química , Ressonância de Plasmônio de Superfície
6.
Eur J Med Chem ; 178: 530-543, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31212132

RESUMO

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 (∼3-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 µM for ERK5; IC50 > 120 µM 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/metabolismo
7.
Mol Cancer Ther ; 6(3): 945-56, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17363489

RESUMO

Poly(ADP-ribose) polymerase (PARP)-1 (EC 2.4.2.30) is a nuclear enzyme that promotes the base excision repair of DNA breaks. Inhibition of PARP-1 enhances the efficacy of DNA alkylating agents, topoisomerase I poisons, and ionizing radiation. Our aim was to identify a PARP inhibitor for clinical trial from a panel of 42 potent PARP inhibitors (K(i), 1.4-15.1 nmol/L) based on the quinazolinone, benzimidazole, tricyclic benzimidazole, tricyclic indole, and tricyclic indole-1-one core structures. We evaluated chemosensitization of temozolomide and topotecan using LoVo and SW620 human colorectal cells; in vitro radiosensitization was measured using LoVo cells, and the enhancement of antitumor activity of temozolomide was evaluated in mice bearing SW620 xenografts. Excellent chemopotentiation and radiopotentiation were observed in vitro, with 17 of the compounds causing a greater temozolomide and topotecan sensitization than the benchmark inhibitor AG14361 and 10 compounds were more potent radiosensitizers than AG14361. In tumor-bearing mice, none of the compounds were toxic when given alone, and the antitumor activity of the PARP inhibitor-temozolomide combinations was unrelated to toxicity. Compounds that were more potent chemosensitizers in vivo than AG14361 were also more potent in vitro, validating in vitro assays as a prescreen. These studies have identified a compound, AG14447, as a PARP inhibitor with outstanding in vivo chemosensitization potency at tolerable doses, which is at least 10 times more potent than the initial lead, AG14361. The phosphate salt of AG14447 (AG014699), which has improved aqueous solubility, has been selected for clinical trial.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Animais , Benzodiazepinas/química , Benzodiazepinas/farmacologia , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos da radiação , Neoplasias Colorretais/radioterapia , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Avaliação Pré-Clínica de Medicamentos , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Raios gama , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Compostos Heterocíclicos com 3 Anéis/química , Humanos , Dose Máxima Tolerável , Camundongos , Camundongos Nus , Poli(ADP-Ribose) Polimerases/metabolismo , Relação Estrutura-Atividade , Temozolomida , Inibidores da Topoisomerase I , Topotecan/farmacologia
8.
Cell Cycle ; 15(4): 506-18, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26959608

RESUMO

ERK5, encoded by MAPK7, has been proposed to play a role in cell proliferation, thus attracting interest as a cancer therapeutic target. While oncogenic RAS or BRAF cause sustained activation of the MEK1/2-ERK1/2 pathway, ERK5 is directly activated by MEK5. It has been proposed that RAS and RAF proteins can also promote ERK5 activation. Here we investigated the interplay between RAS-RAF-MEK-ERK and ERK5 signaling and studied the role of ERK5 in tumor cell proliferation in 2 disease-relevant cell models. We demonstrate that although an inducible form of CRAF (CRAF:ER*) can activate ERK5 in fibroblasts, the response is delayed and reflects feed-forward signaling. Additionally, oncogenic KRAS and BRAF do not activate ERK5 in epithelial cells. Although KRAS and BRAF do not couple directly to MEK5-ERK5, ERK5 signaling might still be permissive for proliferation. However, neither the selective MEK5 inhibitor BIX02189 or ERK5 siRNA inhibited proliferation of colorectal cancer cells harbouring KRAS(G12C/G13D) or BRAF(V600E). Furthermore, there was no additive or synergistic effect observed when BIX02189 was combined with the MEK1/2 inhibitor Selumetinib (AZD6244), suggesting that ERK5 was neither required for proliferation nor a driver of innate resistance to MEK1/2 inhibitors. Finally, even cancer cells with MAPK7 amplification were resistant to BIX02189 and ERK5 siRNA, showing that ERK5 amplification does not confer addiction to ERK5 for cell proliferation. Thus ERK5 signaling is unlikely to play a role in tumor cell proliferation downstream of KRAS or BRAF or in tumor cells with ERK5 amplification. These results have important implications for the role of ERK5 as an anti-cancer drug target.


Assuntos
Neoplasias Colorretais/tratamento farmacológico , Proteína Quinase 7 Ativada por Mitógeno/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Compostos de Anilina/administração & dosagem , Benzimidazóis/administração & dosagem , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Indóis/administração & dosagem , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Mutação , Inibidores de Proteínas Quinases/administração & dosagem , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos
9.
Clin Cancer Res ; 10(3): 881-9, 2004 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-14871963

RESUMO

PURPOSE: Mismatch repair (MMR) deficiency confers resistance to temozolomide, a clinically active DNA-methylating agent. The purpose of the current study was to investigate the reversal mechanism of temozolomide resistance by the potent novel poly(ADP-ribose) polymerase (PARP)-1 inhibitor, AG14361, in MMR-proficient and -deficient cells. EXPERIMENTAL DESIGN: The effects of AG14361, in comparison with the methylguanine DNA methyltransferase inhibitor, benzylguanine, on temozolomide-induced growth inhibition were investigated in matched pairs of MMR-proficient (HCT-Ch3, A2780, and CP70-ch3) and -deficient (HCT116, CP70, and CP70-ch2) cells. RESULTS: AG14361 enhanced temozolomide activity in all MMR-proficient cells (1.5-3.3-fold) but was more effective in MMR-deficient cells (3.7-5.2-fold potentiation), overcoming temozolomide resistance. In contrast, benzylguanine only increased the efficacy of temozolomide in MMR-proficient cells but was ineffective in MMR-deficient cells. The differential effect of AG14361 in MMR-deficient cells was not attributable to differences in PARP-1 activity or differences in its inhibition by AG14361, nor was it attributable to differences in DNA strand breaks induced by temozolomide plus AG14361. MMR-deficient cells are resistant to cisplatin, but AG14361 did not sensitize any cells to cisplatin. PARP-1 inhibitors potentiate topotecan-induced growth inhibition, but AG14361 did not potentiate topotecan in MMR-deficient cells more than in MMR-proficient cells. CONCLUSIONS: MMR defects are relatively common in sporadic tumors and cancer syndromes. PARP-1 inhibition represents a novel way of selectively targeting such tumors. The underlying mechanism is probably a shift of the cytotoxic locus of temozolomide to N(7)-methylguanine and N(3)-methyladenine, which are repaired by the base excision repair pathway in which PARP-1 actively participates.


Assuntos
Pareamento Incorreto de Bases , Benzodiazepinas/farmacologia , Reparo do DNA , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Inibidores Enzimáticos/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Adenina/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos Alquilantes/farmacologia , Azulenos , Divisão Celular , Linhagem Celular Tumoral , Cisplatino/farmacologia , Dano ao DNA , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Genótipo , Guanina/análogos & derivados , Guanina/farmacologia , Humanos , Modelos Químicos , NAD , Temozolomida
10.
Clin Cancer Res ; 9(7): 2711-8, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12855651

RESUMO

The nuclear enzyme poly(ADP-ribose) polymerase (PARP-1) facilitates DNA repair, and is, therefore, an attractive target for anticancer chemo- and radio-potentiation. Novel benzimidazole-4-carboxamides (BZ1-6) and tricyclic lactam indoles (TI1-5) with PARP-1 K(i) values of <10 nM have been identified. Whole cell PARP-1 inhibition, intrinsic cell growth inhibition, and chemopotentiation of the cytotoxic agents temozolomide (TM) and topotecan (TP) were evaluated in LoVo human colon carcinoma cells. The acute toxicity of the inhibitors was investigated in PARP-1 null and wild-type mice. Tissue distribution and in vivo chemopotentiation activity was determined in nude mice bearing LoVo xenografts. At a nontoxic concentration (0.4 micro M) the PARP-1 inhibitors potentiated TM-induced growth inhibition 1.0-5.3-fold and TP-induced inhibition from 1.0-2.1-fold. Concentrations of the PARP-1 inhibitors that alone inhibited cell growth by 50% ranged from 8 to 94 micro M. Maximum potentiation of TM activity was achieved at nongrowth inhibitory concentrations (

Assuntos
Dacarbazina/análogos & derivados , Inibidores Enzimáticos/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Antineoplásicos/farmacologia , Antineoplásicos Alquilantes/farmacologia , Divisão Celular , Linhagem Celular Tumoral , Reparo do DNA , Dacarbazina/uso terapêutico , Dacarbazina/toxicidade , Relação Dose-Resposta a Droga , Humanos , Indóis/metabolismo , Cinética , Modelos Químicos , Poli(ADP-Ribose) Polimerases/genética , Temozolomida , Temperatura , Fatores de Tempo , Distribuição Tecidual , Topotecan/uso terapêutico , Topotecan/toxicidade
11.
J Med Chem ; 47(22): 5467-81, 2004 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-15481984

RESUMO

The design, synthesis, and biological evaluation of potent inhibitors of poly(ADP-ribose) polymerase-1 (PARP-1) are reported. A novel series of 3,4-dihydro-2H-[1,4]diazepino[6,7,1-hi]indol-1-ones were designed using a combination of protein structure-based drug design, molecular modeling, and structure-activity relationships (SAR). These novel submicromolar inhibitors possess a tricyclic ring system conformationally restricting the benzamide in the preferred cis orientation. The compounds were designed to optimize space-filling and atomic interactions within the NAD+ binding site of PARP-1. Previously described and newly adapted methods were applied to syntheses of these tricyclic inhibitors. Various modifications were made to the diazepinoindolones at the 6- and 7-positions in order to study this region of the active site and optimize noncovalent interactions. The electron density of derivative 28 bound to chicken PARP-1 revealed that the oxime makes a tight hydrogen bond with the catalytic gamma-carboxylate of glutamic acid (Glu) 988 in accordance with our original designs and models. Most of the compounds have been evaluated for inhibition of human PARP-1. Selected inhibitors were also tested for the ability to potentiate the cytotoxic effect of the DNA-damaging agent Topotecan.


Assuntos
Antineoplásicos/síntese química , Azepinas/síntese química , Indóis/síntese química , Inibidores de Poli(ADP-Ribose) Polimerases , Antineoplásicos/química , Antineoplásicos/farmacologia , Azepinas/química , Azepinas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Desenho de Fármacos , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Humanos , Indóis/química , Indóis/farmacologia , Modelos Moleculares , Relação Estrutura-Atividade , Inibidores da Topoisomerase I
12.
J Med Chem ; 47(15): 3710-22, 2004 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-15239650

RESUMO

The adenosine 5'-triphosphate (ATP) competitive cyclin-dependent kinase inhibitor O(6)-cyclohexylmethylguanine (NU2058, 1) has been employed as the lead in a structure-based drug discovery program resulting in the discovery of the potent CDK1 and -2 inhibitor NU6102 (3, IC(50) = 9.5 nM and 5.4 nM vs CDK1/cyclinB and CDK2/cyclinA3, respectively). The SAR for this series have been explored further by the synthesis and evaluation of 45 N(2)-substituted analogues of NU2058. These studies have confirmed the requirement for the hydrogen bonding N(2)-NH group and the requirement for an aromatic N(2)-substituent to confer potency in the series. Additional potency is conferred by the presence of a group capable of donating a hydrogen bond at the 4'-position, for example, the 4'-hydroxy derivative (25, IC(50) = 94 nM and 69 nM vs CDK1/cyclinB and CDK2/cyclinA3, respectively), 4'-monomethylsulfonamide derivative (28, IC(50) = 9 nM and 7.0 nM vs CDK1/cyclinB and CDK2/cyclinA3, respectively), and 4'-carboxamide derivative (34, IC(50) = 67 nM and 64 nM vs CDK1/cyclinB and CDK2/cyclinA3, respectively). X-ray crystal structures have been obtained for key compounds and have been used to explain the observed trends in activity.


Assuntos
Proteína Quinase CDC2/antagonistas & inibidores , Quinases relacionadas a CDC2 e CDC28/antagonistas & inibidores , Cicloexanos/síntese química , Guanina/análogos & derivados , Guanina/síntese química , Purinas/síntese química , Animais , Proteína Quinase CDC2/química , Quinases relacionadas a CDC2 e CDC28/química , Cristalografia por Raios X , Quinase 2 Dependente de Ciclina , Cicloexanos/química , Guanina/química , Humanos , Modelos Moleculares , Purinas/química , Purinas/farmacologia , Estrelas-do-Mar , Relação Estrutura-Atividade
13.
J Med Chem ; 45(23): 4961-74, 2002 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-12408707

RESUMO

A series of novel compounds have been designed that are potent inhibitors of poly(ADP-ribose) polymerase-1 (PARP-1), and the activity and physical properties have been characterized. The new structural classes, 3,4,5,6-tetrahydro-1H-azepino[5,4,3-cd]indol-6-ones and 3,4-dihydropyrrolo[4,3,2-de]isoquinolin-5-(1H)-ones, have conformationally locked benzamide cores that specifically interact with the PARP-1 protein. The compounds have been evaluated with in vitro cellular assays that measure the ability of the PARP-1 inhibitors to enhance the effect of cytotoxic agents against cancer cell lines.


Assuntos
Antineoplásicos/síntese química , Dacarbazina/análogos & derivados , Inibidores Enzimáticos/síntese química , Indóis/síntese química , Isoquinolinas/síntese química , Inibidores de Poli(ADP-Ribose) Polimerases , Antineoplásicos/química , Antineoplásicos/farmacologia , Cristalografia por Raios X , Dacarbazina/farmacologia , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Indóis/química , Indóis/farmacologia , Isoquinolinas/química , Isoquinolinas/farmacologia , NAD/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Relação Estrutura-Atividade , Temozolomida , Topotecan/farmacologia , Células Tumorais Cultivadas
14.
Eur J Med Chem ; 68: 333-8, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23994326

RESUMO

Substitution of the cyano-NNO-azoxy moiety (NC-N=(O)N-) for the nitroso group in NU6027, a potent and selective CDK2 inhibitor, affords a compound with slightly improved potency and comparable selectivity profile. A molecular modelling study indicates for this new scaffold a binding mode similar to the one adopted by other purine and pyrimidine analogues, and suggests a relevant role for a conserved water molecule in stabilizing the bioactive pose of this and other pyrimidine ligands. The introduction of aminosulfonylphenyl substituents on the 2-amino group of the pyrimidine increased the CDK2 inhibitory potency by two orders of magnitude, while maintaining the same degree of selectivity.


Assuntos
Aminas/química , Aminas/farmacologia , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Ativação Enzimática/efeitos dos fármacos , Concentração Inibidora 50 , Modelos Moleculares , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Relação Estrutura-Atividade
15.
Structure ; 20(10): 1788-95, 2012 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-22959624

RESUMO

CDK9, the kinase of positive transcription elongation factor b (P-TEFb), stimulates transcription elongation by phosphorylating RNA polymerase II and transcription elongation factors. Using kinetic analysis of a human P-TEFb complex consisting of CDK9 and cyclin T, we show that the CDK9 C-terminal tail sequence is important for the catalytic mechanism and imposes an ordered binding of substrates and release of products. Crystallographic analysis of a CDK9/cyclin T complex in which the C-terminal tail partially blocks the ATP binding site reveals a possible reaction intermediate. Biochemical characterization of CDK9 mutants supports a model in which the CDK9 tail cycles through different conformational states. We propose that this mechanism is critical for the pattern of CTD Ser2 phosphorylation on actively transcribed genes.


Assuntos
Ciclina T/química , Quinase 9 Dependente de Ciclina/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Quinase 9 Dependente de Ciclina/genética , Diclororribofuranosilbenzimidazol/química , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/química , Fator B de Elongação Transcricional Positiva/química , Ligação Proteica , Inibidores de Proteínas Quinases/química
16.
Eur J Cancer ; 47(13): 2052-9, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21570822

RESUMO

To facilitate the evaluation of CDK2 (cyclin-dependent kinase 2) as a cancer target, the in vitro and in vivo properties of NU6102 (O6-cyclohexylmethyl-2-(4'-sulphamoylanilino)purine) and a water soluble prodrug (NU6301) were investigated. NU6102 selectively inhibited the growth of CDK2 WT (wild type) versus KO MEFs (knockout mouse embryo fibroblasts) (GI50 (concentration required to inhibit cell growth by 50%) 14 µM versus >30 µM), and was more growth-inhibitory in p53 mutant or null versus p53 WT cells (p=0.02), and in Rb (retinoblastoma protein) WT SKUT-1B versus SKUT 1 Rb deficient cells (p=0.01). In SKUT-1B cells NU6102 induced a G2 arrest, inhibition of Rb phosphorylation and cytotoxicity (LC50 2.6 µM for a 24h exposure). The prodrug NU6301 rapidly generated NU6102 in vitro in mouse plasma, and tumour NU6102 levels in vivo consistent with activity in vitro. Eight or 12 hourly dosing of 120 mg/kg NU6301 for 10 days was well tolerated in SKUT-1B tumour-bearing mice and inhibited Rb phosphorylation in tumour tissue. Two (8 hourly dosing) and 3 (12 hourly dosing) day tumour growth delay was observed (p=0.04 and p=0.007, respectively) following NU6301 administration. NU6102 and its prodrug NU6301 have pharmacological properties consistent with CDK2 inhibition, and represent useful tool molecules for the evaluation of CDK2 as a target in cancer.


Assuntos
Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Pró-Fármacos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Purinas/farmacologia , Animais , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Células HCT116 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Pró-Fármacos/farmacocinética , Inibidores de Proteínas Quinases/farmacocinética , Purinas/farmacocinética , Proteína do Retinoblastoma/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Mol Cancer Ther ; 8(7): 1828-37, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19509274

RESUMO

Antifolates have been used to treat cancer for the last 50 years and remain the mainstay of many therapeutic regimes. Nucleoside salvage, which depends on plasma membrane transport, can compromise the activity of antifolates. The cardiovascular drug dipyridamole inhibits nucleoside transport and enhances antifolate cytotoxicity in vitro, but its clinical activity is compromised by binding to the plasma protein alpha(1)-acid glycoprotein (AGP). We report the development of a novel pyrimidopyrimidine analogue of dipyridamole, NU3153, which has equivalent potency to dipyridamole, remains active in the presence of physiologic levels of AGP, inhibits thymidine incorporation into DNA, and prevents thymidine and hypoxanthine rescue from the multitargeted antifolate, pemetrexed. Pharmacokinetic evaluation of NU3153 suggested that a soluble prodrug would improve the in vivo activity. The valine prodrug of NU3153, NU3166, rapidly broke down to NU3153 in vitro and in vivo. Plasma NU3153 concentrations commensurate with rescue inhibition in vitro were maintained for at least 16 hours following administration of NU3166 to mice at 120 mg/kg. However, maximum inhibition of thymidine incorporation into tumors was only 50%, which was insufficient to enhance pemetrexed antitumor activity in vivo. Comparison with the cell-based studies revealed that pemetrexed enhancement requires substantial (> or =90%) and durable inhibition of nucleoside transport. In conclusion, we have developed non-AGP binding nucleoside transport inhibitors. Pharmacologically active concentrations of the inhibitors can be achieved in vivo using prodrug approaches, but greater potency is required to evaluate inhibition of nucleoside rescue as a therapeutic maneuver.


Assuntos
Dipiridamol/análogos & derivados , Antagonistas do Ácido Fólico/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Nucleosídeos/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Antimetabólitos Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Dipiridamol/farmacocinética , Dipiridamol/farmacologia , Sinergismo Farmacológico , Feminino , Antagonistas do Ácido Fólico/farmacocinética , Glutamatos/farmacologia , Guanina/análogos & derivados , Guanina/farmacologia , Humanos , Leucemia L1210/tratamento farmacológico , Leucemia L1210/metabolismo , Leucemia L1210/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Orosomucoide/metabolismo , Pemetrexede , Fosforilação/efeitos dos fármacos , Pró-Fármacos/farmacocinética , Pró-Fármacos/farmacologia , Timidina/metabolismo , Timidilato Sintase/antagonistas & inibidores , Distribuição Tecidual , Células Tumorais Cultivadas
18.
Cell Cycle ; 7(24): 3898-907, 2008 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-19066469

RESUMO

Transient treatment with small molecule CDK inhibitors is toxic to cancer cells and leads to depletion of anti-apoptotic proteins and Chk1, coupled with DNA damage and induction of apoptosis. Here we have examined, which of these phenomena are necessary for CDK inhibitors to have an anti-proliferative effect. We find that 24 hours treatment with either a primarily CDK2-specific, or a primarily CDK7/9-specific, antagonist eliminates proliferative potential even if apoptosis is blocked and the tendency of CDK inhibition to result in DNA damage is overcome by expression of recombinant Chk1. Loss of proliferative potential is correlated with irreversible suppression of biomarkers of cell cycle progression. CDK inhibitors dramatically reduced levels of the anti-apoptotic proteins, Mcl-1 and XIAP, but siRNA-mediated suppression of Mcl-1 and XIAP did not induce cell death in the osteosarcoma cells used in this study. Finally, we found that many literature CDK inhibitors do not effectively suppress the CDK/cyclin complexes responsible for cell cycle progression at the minimum doses required to block proliferation: some are only effective after a substantial delay and may act via inhibition of CDK7.


Assuntos
Antineoplásicos/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Quinase 1 do Ponto de Checagem , Quinases Ciclina-Dependentes/metabolismo , Dano ao DNA , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Oxazóis/farmacologia , Proteínas Quinases/biossíntese , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , RNA Interferente Pequeno , Tiazóis/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
19.
J Am Chem Soc ; 128(18): 6012-3, 2006 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-16669651

RESUMO

beta-Piperidinoethylsulfides are oxidized by m-chloroperbenzoic acid to intermediates containing both N-oxide and sulfone functions. These undergo a Cope-type elimination to a vinylsulfone that can be captured by amines to afford beta-aminoethylsulfones. When a beta-aminoethylsulfone group is linked to the 4-position of a phenyl group attached at N-2 of O6-cyclohexylmethylguanine, the resulting derivatives are inhibitors of the cyclin-dependent kinase CDK2. One of the most potent inhibitors (IC50 = 45 nM) contained a N-3-hydroxypropyl group on the aminoethylsulfonyl substituent. The crystal structure of this inhibitor bound to CDK2/cyclin A was determined and shows an unusual network of hydrogen bonds. The synthetic methodology developed can be utilized in multiple-parallel format and has numerous potential applications in medicinal chemistry.


Assuntos
Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Sulfonamidas/química , Sulfonamidas/farmacologia , Sulfonas/química , Sulfonas/farmacologia , Cristalografia por Raios X , Quinase 2 Dependente de Ciclina/química , Desenho de Fármacos , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonas/síntese química
20.
J Pharmacol Exp Ther ; 312(1): 206-13, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15304523

RESUMO

Pharmacological inhibitors of cyclin-dependent kinase (CDK)2 are currently in preclinical and clinical development. The purpose of our work was to evaluate a series of guanine derivatives for their ability to inhibit CDK2, affect cell cycle progression, and enhance the cytotoxic and apoptotic effects of cisplatin. A panel of guanine derivatives, including O(6)-benzylguanine (O(6)-BG), S(6)-benzyl-6-thioguanine (S(6)-BG), S(6)-[(cyclohexyl)methyl]-6-thioguanine (S(6)-CMG), O(6)-[(cyclohexyl)methyl]guanine (O(6)-CMG), O(6)-benzyl-9-methylguanine (9-CH(3)-BG), O(6)-[(cyclohexyl)methyl]-9-methyl-guanine (9-CH(3)-CMG), and 7-benzylguanine (N7-BG), exhibited varying degrees of CDK2 inhibition with O(6)-CMG being the most potent and 9-CH(3)-BG, 9-CH(3)-CMG, and N7-BG the least potent compounds. Treatment with S(6)-CMG and O(6)-CMG significantly decreased the percentage of cells in S phase. In SQ20b and SCC61 head and neck cancer cell lines, the most potent CDK2 inhibitor, O(6)-CMG, was also the most effective at enhancing cisplatin-induced cytotoxicity and apoptosis. Cisplatin-induced DNA platination increased in SQ20b cells pretreated with S(6)-BG, S(6)-CMG, and O(6)-CMG. Treatment with both O(6)-BG and trichostatin A, an indirect cell cycle inhibitor, demonstrated additive effects on cisplatin-induced cytotoxicity. In summary, we have identified a group of guanine derivatives that were effective modulators of cisplatin-induced cytotoxicity and apoptosis.


Assuntos
Antineoplásicos/farmacologia , Quinases relacionadas a CDC2 e CDC28/antagonistas & inibidores , Ciclo Celular/efeitos dos fármacos , Cisplatino/farmacologia , Guanina/análogos & derivados , Guanina/farmacologia , Quinase 2 Dependente de Ciclina , DNA/efeitos dos fármacos , DNA/metabolismo , Combinação de Medicamentos , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , O(6)-Metilguanina-DNA Metiltransferase/antagonistas & inibidores , Células Tumorais Cultivadas
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