RESUMO
The p53 tumor suppressor is controlled by MDM2, which binds p53 and negatively regulates its transcriptional activity and stability. Many tumors overproduce MDM2 to impair p53 function. Therefore, restoration of p53 activity by inhibiting the p53-MDM2 binding represents an attractive novel approach to cancer therapy. Recently developed potent and selective small-molecule antagonists of the p53-MDM2 interaction have been used to demonstrate the proof-of-concept for this approach. These compounds interact specifically with the p53-binding pocket of MDM2 and release p53 from negative control. Treatment of cancer cells expressing wild-type p53 stabilize p53 and activate the p53 pathway, leading to cell cycle arrest and apoptosis. In mice-bearing established human tumor xenografts, MDM2 antagonists caused tumor inhibition and regression at nontoxic concentrations, suggesting that they may have a therapeutic utility in the treatment of cancer. An increasing number of MDM2 antagonists are being generated and some of them have entered clinical trials. Here, we review this class of emerging drugs with an emphasis on small molecules that inhibit the p53-MDM2 interaction.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Sítios de Ligação , Biomarcadores Tumorais/metabolismo , Descoberta de Drogas/métodos , Inibidores Enzimáticos/farmacologia , Humanos , Modelos Moleculares , Terapia de Alvo Molecular , Ligação Proteica/efeitos dos fármacosRESUMO
Recent studies have shown that activation of cell cycle checkpoints can protect normal proliferating cells from mitotic inhibitors by preventing their entry into mitosis. These studies have used genotoxic agents that act, at least in part, by activation of the p53 pathway. However, genotoxic drugs are known also to have p53-independent activities and could affect the sensitivity of tumor cells to antimitotic agents. Recently, we have developed the first potent and selective small-molecule inhibitors of the p53-MDM2 interaction, the nutlins, which activate the p53 pathway only in cells with wild-type but not mutant p53. Using these compounds, we show that p53 activation leads to G1 and G2 phase arrest and can protect cells from mitotic block and apoptosis caused by paclitaxel. Pretreatment of HCT116 and RKO colon cancer cells (wild-type p53) or primary human fibroblasts (1043SK) with nutlins for 24 hours followed by incubation with paclitaxel for additional 48 hours did not increase significantly their mitotic index and protected the cells from the cytotoxicity of paclitaxel. Cancer cells with mutant p53 (MDA-MB-435) responded to the same treatment with mitotic arrest and massive apoptosis. These results have two major implications for cancer therapy. First, p53-activating therapies may have antagonistic effect when combined with mitotic poisons. Second, pretreatment with MDM2 antagonists before chemotherapy of tumors with mutant p53 may offer a partial protection to proliferating normal tissues.
Assuntos
Neoplasias da Mama/metabolismo , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Imidazóis/farmacologia , Proteínas Nucleares/antagonistas & inibidores , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Antineoplásicos Fitogênicos/efeitos adversos , Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Neoplasias do Colo/patologia , Feminino , Fase G1/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Humanos , Mitose/efeitos dos fármacos , Mutação/genética , Proteínas Nucleares/metabolismo , Paclitaxel/efeitos adversos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2 , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
The cyclin-dependent kinases (CDKs) and their cyclin partners are key regulators of the cell cycle. Since deregulation of CDKs is found with high frequency in many human cancer cells, pharmacological inhibition of CDKs with small molecules has the potential to provide an effective strategy for the treatment of cancer. The 2,4-diamino-5-ketopyrimidines 6 reported here represent a novel class of potent and ATP-competitive inhibitors that selectively target the cyclin-dependent kinase family. This diaminopyrimidine core with a substituted 4-piperidine moiety on the C2-amino position and 2-methoxybenzoyl at the C5 position has been identified as the critical structure responsible for the CDK inhibitory activity. Further optimization has led to a good number of analogues that show potent inhibitory activities against CDK1, CDK2, and CDK4 but are inactive against a large panel of serine/threonine and tyrosine kinases (K(i) > 10 microM). As one of these representative analogues, compound 39 (R547) has the best CDK inhibitory activities (K(i) = 0.001, 0.003, and 0.001 microM for CDK1, CDK2, and CDK4, respectively) and excellent in vitro cellular potency, inhibiting the growth of various human tumor cell lines including an HCT116 cell line (IC(50) = 0.08 microM). An X-ray crystal structure of 39 bound to CDK2 has been determined in this study, revealing a binding mode that is consistent with our SAR. Compound 39 demonstrates significant in vivo efficacy in the HCT116 human colorectal tumor xenograft model in nude mice with up to 95% tumor growth inhibition. On the basis of its superior overall profile, 39 was chosen for further evaluation and has progressed into Phase I clinical trial for the treatment of cancer.
Assuntos
Antineoplásicos/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/farmacocinética , Pirimidinas/farmacologia , Pirimidinas/farmacocinética , Animais , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Fenômenos Químicos , Físico-Química , Quinase 2 Dependente de Ciclina/química , Desenho de Fármacos , Feminino , Humanos , Indicadores e Reagentes , Cinética , Camundongos , Camundongos Nus , Modelos Moleculares , Relação Estrutura-Atividade , Difração de Raios XRESUMO
The diastereoselectivity of intramolecular 1,3-dipolar cycloadditions of 2-substituted 5-hexenyl and 5-heptenyl nitrones to give 6-substituted and 3,6-disubstituted perhydrocyclopenta[c]isoxazoles has been investigated. An alkyl or aryl substituent at C2 completely controls the stereochemistry of the ring juncture and, in the case of the 5-heptenyl systems, also the stereochemistry of the 3-methyl group. Thus one stereocenter controls the formation of the other three to give a product with four contiguous stereocenters. The use of an ethylene ketal substituent in these systems allows the reaction to be carried out at much lower temperatures, an example of the gem-dialkoxy effect. This cycloaddition process has been used in an efficient formal total synthesis of the potent beta-lactam antibiotic, 1beta-methylthienamycin.
RESUMO
The p53 tumor suppressor retains its wild-type conformation and transcriptional activity in half of all human tumors, and its activation may offer a therapeutic benefit. However, p53 function could be compromised by defective signaling in the p53 pathway. Using a small-molecule MDM2 antagonist, nutlin-3, to probe downstream p53 signaling we find that the cell-cycle arrest function of the p53 pathway is preserved in multiple tumor-derived cell lines expressing wild-type p53, but many have a reduced ability to undergo p53-dependent apoptosis. Gene array analysis revealed attenuated expression of multiple apoptosis-related genes. Cancer cells with mdm2 gene amplification were most sensitive to nutlin-3 in vitro and in vivo, suggesting that MDM2 overexpression may be the only abnormality in the p53 pathway of these cells. Nutlin-3 also showed good efficacy against tumors with normal MDM2 expression, suggesting that many of the patients with wild-type p53 tumors may benefit from antagonists of the p53-MDM2 interaction.
Assuntos
Antineoplásicos/farmacologia , Imidazóis/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Nus , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
MDM2 is a regulator of cell growth processes that acts by binding to the tumor suppressor protein p53 and ultimately restraining its activity. While inactivation of p53 by mutation is commonly observed in human cancers, a substantial percentage of tumors express wild type p53. In many of these cases, MDM2 is overexpressed, and it is believed that suppression of MDM2 activity could yield therapeutic benefits. Therefore, we have been focusing on the p53-MDM2 interaction as the basis of a drug discovery program and have been able to develop a series of small molecule inhibitors. We herein report a high resolution NMR structure of a complex between the p53-binding domain of MDM2 and one of these inhibitors. The form of MDM2 utilized was an engineered hybrid between the human and Xenopus sequences, which provided a favorable combination of relevancy and stability. The inhibitor is found to bind in the same site as does a highly potent peptide fragment of p53. The inhibitor is able to successfully mimic the peptide by duplicating interactions in three subpockets normally made by amino acid sidechains, and by utilizing a scaffold that presents substituents with rigidity and spatial orientation comparable to that provided by the alpha helical backbone of the peptide. The structure also suggests opportunities for modifying the inhibitor to increase its potency.
Assuntos
Ressonância Magnética Nuclear Biomolecular , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Desenho de Fármacos , Humanos , Ligantes , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Xenopus laevisRESUMO
The p53 tumor suppressor is a key mediator of the cellular response to stress. Phosphorylation induced by multiple stress-activated kinases has been proposed to be essential for p53 stabilization, interaction with transcriptional co-activators, and activation of p53 target genes. However, genetic studies suggest that stress-activated phosphorylation may not be essential for p53 activation. We therefore investigated the role of p53 phosphorylation on six key serine residues (Ser(6), Ser(15), Ser(20), Ser(37), Ser(46), and Ser(392)) for p53 activation using nutlin-3, a recently developed small molecule MDM2 antagonist. We show here that nutlin does not induce the phosphorylation of p53. Comparison of the activity of unphosphorylated and phosphorylated p53 induced by the genotoxic drugs doxorubicin and etoposide in HCT116 and RKO cells revealed no difference in their sequence-specific DNA binding and ability to transactivate p53 target genes and to induce p53-dependent apoptosis. We conclude that p53 phosphorylation on six major serine sites is not required for activation of p53 target genes or biological responses in vivo.
Assuntos
Apoptose , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismo , Anexina A5/farmacologia , Antibióticos Antineoplásicos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Western Blotting , Linhagem Celular Tumoral , Corantes/farmacologia , DNA/química , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Ensaio de Imunoadsorção Enzimática , Etoposídeo/farmacologia , Humanos , Imidazóis/metabolismo , Cinética , Proteínas Nucleares/metabolismo , Fosforilação , Piperazinas/metabolismo , Reação em Cadeia da Polimerase , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2 , Serina/químicaRESUMO
MDM2 binds the p53 tumor suppressor protein with high affinity and negatively modulates its transcriptional activity and stability. Overexpression of MDM2, found in many human tumors, effectively impairs p53 function. Inhibition of MDM2-p53 interaction can stabilize p53 and may offer a novel strategy for cancer therapy. Here, we identify potent and selective small-molecule antagonists of MDM2 and confirm their mode of action through the crystal structures of complexes. These compounds bind MDM2 in the p53-binding pocket and activate the p53 pathway in cancer cells, leading to cell cycle arrest, apoptosis, and growth inhibition of human tumor xenografts in nude mice.