RESUMO
First line treatment for pancreatic cancer consists of surgical resection, if possible, and a subsequent course of chemotherapy using the nucleoside analogue gemcitabine. In some patients, an active transport mechanism allows gemcitabine to enter efficiently into the tumor cells, resulting in a significant clinical benefit. However, in most patients, low expression of gemcitabine transporters limits the efficacy of the drug to marginal levels, and patients need frequent administration of the drug at high doses, significantly increasing systemic drug toxicity. In this article we focus on a novel targeted delivery approach for gemcitabine consisting of conjugating the drug with an EphA2 targeting agent. We show that the EphA2 receptor is highly expressed in pancreatic cancers, and accordingly, the drug-conjugate is more effective than gemcitabine alone in targeting pancreatic tumors. Our preliminary observations suggest that this approach may provide a general benefit to pancreatic cancer patients and offers a comprehensive strategy for enhancing delivery of diverse therapeutic agents to a wide range of cancers overexpressing EphA2, thereby potentially reducing toxicity while enhancing therapeutic efficacy.
Assuntos
Antimetabólitos Antineoplásicos/administração & dosagem , Desoxicitidina/análogos & derivados , Terapia de Alvo Molecular/métodos , Neoplasias Pancreáticas/tratamento farmacológico , Receptor EphA2/metabolismo , Animais , Linhagem Celular Tumoral , Desoxicitidina/administração & dosagem , Humanos , Camundongos , Camundongos Nus , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
The development of novel, targeted delivery agents for anti-cancer therapies requires the design and optimization of potent and selective tumor-targeting agents that are stable and amenable to conjugation with chemotherapeutic drugs. While short peptides represent potentially an excellent platform for these purposes, they often get degraded and are eliminated too rapidly in vivo. In this study, we used a combination of nuclear magnetic resonance-guided structure-activity relationships along with biochemical and cellular studies to derive a novel tumor-homing agent, named 123B9, targeting the EphA2 tyrosine kinase receptor ligand-binding domain. Conjugating 123B9 to the chemotherapeutic drug paclitaxel (PTX) via a stable linker results in an agent that is significantly more effective than the unconjugated drug in both a pancreatic cancer xenograft model and a melanoma lung colonization and metastases model. Hence, 123B9 could represent a promising strategy for the development of novel targeted therapies for cancer.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Paclitaxel/análogos & derivados , Receptor EphA2/agonistas , Sequência de Aminoácidos , Animais , Antineoplásicos/síntese química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sistemas de Liberação de Medicamentos/métodos , Feminino , Humanos , Masculino , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/metabolismo , Camundongos , Camundongos Nus , Modelos Animais , Terapia de Alvo Molecular , Paclitaxel/química , Paclitaxel/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Ratos , Receptor EphA2/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Because of its overexpression in a range of solid tumors, the EphA2 receptor is a validated target for cancer therapeutics. We recently described a new targeted delivery system based on specific EphA2-targeting peptides conjugated with the chemotherapeutic agent paclitaxel. Here, we investigate the chemical determinants responsible for the stability and degradation of these agents in plasma. Introducing modifications in both the peptide and the linker between the peptide and paclitaxel resulted in drug conjugates that are both long-lived in rat plasma and that markedly decrease tumor size in a prostate cancer xenograft model compared with paclitaxel alone treatment. These studies identify critical rate-limiting degradation sites on the peptide-drug conjugates, enabling the design of agents with increased stability and efficacy. These results provide support for our central hypothesis that peptide-drug conjugates targeting EphA2 represent an innovative and potentially effective strategy to selectively deliver cytotoxic drugs to cancer cells.
Assuntos
Portadores de Fármacos/síntese química , Desenho de Fármacos , Receptor EphA2/metabolismo , Sequência de Aminoácidos , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Portadores de Fármacos/química , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Paclitaxel/química , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Peptídeos/síntese química , Peptídeos/química , Neoplasias da Próstata/tratamento farmacológico , Receptor EphA2/química , Transplante HeterólogoRESUMO
The E3 ubiquitin ligase Siah regulates key cellular events that are central to cancer development and progression. A promising route to Siah inhibition is disrupting its interactions with adaptor proteins. However, typical of protein-protein interactions, traditional unbiased approaches to ligand discovery did not produce viable hits against this target, despite considerable effort and a multitude of approaches. Ultimately, a rational structure-based design strategy was successful for the identification of Siah inhibitors in which peptide binding drives specific covalent bond formation with the target. X-ray crystallography, mass spectrometry, and functional data demonstrate that these peptide mimetics are efficient covalent inhibitors of Siah and antagonize Siah-dependent regulation of Erk and Hif signaling in the cell. The proposed strategy may result useful as a general approach to the design of peptide-based inhibitors of other protein-protein interactions.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/química , Proteínas Nucleares/antagonistas & inibidores , Peptídeos/química , Peptidomiméticos/química , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Peptídeos/farmacologia , Peptidomiméticos/farmacologia , Mapas de Interação de Proteínas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismoRESUMO
PURPOSE: YSA is an EphA2-targeting peptide that effectively delivers anticancer agents to prostate cancer tumors. Here, we report on how we increased the drug-like properties of this delivery system. EXPERIMENTAL DESIGN: By introducing non-natural amino acids, we have designed two new EphA2 targeting peptides: YNH, where norleucine and homoserine replace the two methionine residues of YSA, and dYNH, where a D-tyrosine replaces the L-tyrosine at the first position of the YNH peptide. We describe the details of the synthesis of YNH and dYNH paclitaxel conjugates (YNH-PTX and dYNH-PTX) and their characterization in cells and in vivo. RESULTS: dYNH-PTX showed improved stability in mouse serum and significantly reduced tumor size in a prostate cancer xenograft model and also reduced tumor vasculature in a syngeneic orthotopic allograft mouse model of renal cancer compared with vehicle or paclitaxel treatments. CONCLUSION: This study reveals that targeting EphA2 with dYNH drug conjugates could represent an effective way to deliver anticancer agents to a variety of tumor types.
Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Sistemas de Liberação de Medicamentos , Neoplasias/genética , Paclitaxel/administração & dosagem , Peptídeos , Receptor EphA2/genética , Animais , Antineoplásicos Fitogênicos/química , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Masculino , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/genética , Paclitaxel/química , Peptídeos/química , Receptor EphA2/metabolismo , Transplante Homólogo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Structure-based modeling combined with rational drug design, and high throughput screening approaches offer significant potential for identifying and developing lead compounds with therapeutic potential. The present review focuses on these two approaches using explicit examples based on specific derivatives of Gossypol generated through rational design and applications of a cancer-specificpromoter derived from Progression Elevated Gene-3. The Gossypol derivative Sabutoclax (BI-97C1) displays potent anti-tumor activity against a diverse spectrum of human tumors. The model of the docked structure of Gossypol bound to Bcl-XL provided a virtual structure-activity-relationship where appropriate modifications were predicted on a rational basis. These structure-based studies led to the isolation of Sabutoclax, an optically pure isomer of Apogossypol displaying superior efficacy and reduced toxicity. These studies illustrate the power of combining structure-based modeling with rational design to predict appropriate derivatives of lead compounds to be empirically tested and evaluated for bioactivity. Another approach to cancer drug discovery utilizes a cancer-specific promoter as readouts of the transformed state. The promoter region of Progression Elevated Gene-3 is such a promoter with cancer-specific activity. The specificity of this promoter has been exploited as a means of constructing cancer terminator viruses that selectively kill cancer cells and as a systemic imaging modality that specifically visualizes in vivo cancer growth with no background from normal tissues. Screening of small molecule inhibitors that suppress the Progression Elevated Gene-3-promoter may provide relevant lead compounds for cancer therapy that can be combined with further structure-based approaches leading to the development of novel compounds for cancer therapy.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Gossipol/análogos & derivados , Gossipol/farmacologia , Neoplasias/tratamento farmacológico , Animais , Ensaios de Seleção de Medicamentos Antitumorais/economia , Ensaios de Triagem em Larga Escala , Humanos , Neoplasias/genética , Regiões Promotoras Genéticas/efeitos dos fármacosRESUMO
Membrane type-1 matrix metalloproteinase (MT1-MMP) is a promising drug target in malignancy. The structure of MT1-MMP includes the hemopexin domain (PEX) that is distinct from and additional to the catalytic domain. Current MMP inhibitors target the conserved active site in the catalytic domain and, as a result, repress the proteolytic activity of multiple MMPs instead of MT1-MMP alone. In our search for noncatalytic inhibitors of MT1-MMP, we compared the protumorigenic activity of wild-type MT1-MMP with an MT1-MMP mutant lacking PEX (ΔPEX). In contrast to MT1-MMP, ΔPEX did not support tumor growth in vivo, and its expression resulted in small fibrotic tumors that contained increased levels of collagen. Because these findings suggested an important role for PEX in tumor growth, we carried out an inhibitor screen to identify small molecules targeting the PEX domain of MT1-MMP. Using the Developmental Therapeutics Program (National Cancer Institute/NIH), virtual ligand screening compound library as a source and the X-ray crystal structure of PEX as a target, we identified and validated a novel PEX inhibitor. Low dosage, intratumoral injections of PEX inhibitor repressed tumor growth and caused a fibrotic, ΔPEX-like tumor phenotype in vivo. Together, our findings provide a preclinical proof of principle rationale for the development of novel and selective MT1-MMP inhibitors that specifically target the PEX domain.
Assuntos
Hemopexina/química , Inibidores de Metaloproteinases de Matriz , Inibidores de Proteases/farmacologia , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/fisiologia , Feminino , Humanos , Metaloproteinase 14 da Matriz/biossíntese , Metaloproteinase 14 da Matriz/química , Metaloproteinase 14 da Matriz/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Moleculares , Estrutura Terciária de Proteína , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The efficacy of anticancer drugs is often limited by their systemic toxicities and adverse side effects. We report that the EphA2 receptor is overexpressed preferentially in several human cancer cell lines compared to normal tissues and that an EphA2 targeting peptide (YSAYPDSVPMMS) can be effective in delivering anticancer agents to such tumors. Hence, we report on the synthesis and characterizations of a novel EphA2-targeting agent conjugated with the chemotherapeutic drug paclitaxel. We found that the peptide-drug conjugate is dramatically more effective than paclitaxel alone at inhibiting tumor growth in a prostate cancer xenograft model, delivering significantly higher levels of drug to the tumor site. We believe these studies open the way to the development of a new class of therapeutic compounds that exploit the EphA2 receptor for drug delivery to cancer cells.
Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Oligopeptídeos/síntese química , Paclitaxel/administração & dosagem , Receptor EphA2/metabolismo , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacocinética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Células Endoteliais/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Camundongos , Camundongos SCID , Transplante de Neoplasias , Oligopeptídeos/química , Paclitaxel/química , Paclitaxel/farmacocinética , RNA Mensageiro/metabolismo , Receptor EphA2/genética , Transplante HeterólogoRESUMO
c-Jun N-terminal kinases (JNKs) represent valuable targets in the development of new therapies. Present on the surface of JNK is a binding pocket for substrates and the scaffolding protein JIP1 in close proximity to the ATP binding pocket. We propose that bidentate compounds linking the binding energies of weakly interacting ATP and substrate mimetics could result in potent and selective JNK inhibitors. We describe here a bidentate molecule, 19, designed against JNK. 19 inhibits JNK kinase activity (IC(50) = 18 nM; K(i) = 1.5 nM) and JNK/substrate association in a displacement assay (IC(50) = 46 nM; K(i) = 2 nM). Our data demonstrate that 19 targets for the ATP and substrate-binding sites on JNK concurrently. Finally, compound 19 successfully inhibits JNK in a variety of cell-based experiments, as well as in vivo where it is shown to protect against Jo-2 induced liver damage and improve glucose tolerance in diabetic mice.
Assuntos
Trifosfato de Adenosina/química , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Peptídeos/química , Motivos de Aminoácidos , Animais , Ligação Competitiva , Linhagem Celular , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Sequência Consenso , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Desenho de Fármacos , Feminino , Intolerância à Glucose/tratamento farmacológico , Humanos , Hipoglicemiantes/síntese química , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/química , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Mimetismo Molecular , Peptídeos/síntese química , Peptídeos/farmacologia , Fosforilação , Ligação Proteica , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
INTRODUCTION: Human cancers are genetically and epigenetically heterogeneous and have the capacity to commandeer a variety of cellular processes to aid in their survival, growth and resistance to therapy. One strategy is to overexpress proteins that suppress apoptosis, such as the Bcl-2 family protein Mcl-1. The Mcl-1 protein plays a pivotal role in protecting cells from apoptosis and is overexpressed in a variety of human cancers. AREAS COVERED: Targeting Mcl-1 for extinction in these cancers, using genetic and pharmacological approaches, represents a potentially effectual means of developing new efficacious cancer therapeutics. Here we review the multiple strategies that have been employed in targeting this fundamental protein, as well as the significant potential these targeting agents provide in not only suppressing cancer growth, but also in reversing resistance to conventional cancer treatments. EXPERT OPINION: We discuss the potential issues that arise in targeting Mcl-1 and other Bcl-2 anti-apoptotic proteins, as well problems with acquired resistance. The application of combinatorial approaches that involve inhibiting Mcl-1 and manipulation of additional signaling pathways to enhance therapeutic outcomes is also highlighted. The ability to specifically inhibit key genetic/epigenetic elements and biochemical pathways that maintain the tumor state represent a viable approach for developing rationally based, effective cancer therapies.
Assuntos
Antineoplásicos/farmacologia , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Animais , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica , Desenho de Fármacos , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Neoplasias/metabolismo , Neoplasias/fisiopatologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Limited options are available for treating patients with advanced prostate cancer (PC). Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24), an IL-10 family cytokine, exhibits pleiotropic anticancer activities without adversely affecting normal cells. We previously demonstrated that suppression of the prosurvival Bcl-2 family member, myeloid cell leukemia-1 (Mcl-1), is required for mda-7/IL-24-mediated apoptosis of prostate carcinomas. Here we demonstrate that pharmacological inhibition of Mcl-1 expression with the unique Apogossypol derivative BI-97C1, also called Sabutoclax, is sufficient to sensitize prostate tumors to mda-7/IL-24-induced apoptosis, whereas ABT-737, which lacks efficacy in inhibiting Mcl-1, does not sensitize mda-7/IL-24-mediated cytotoxicity. A combination regimen of tropism-modified adenovirus delivered mda-7/IL-24 (Ad.5/3-mda-7) and BI-97C1 enhances cytotoxicity in human PC cells, including those resistant to mda-7/IL-24 or BI-97C1 alone. The combination regimen causes autophagy that facilitates NOXA- and Bim-induced and Bak/Bax-mediated mitochondrial apoptosis. Treatment with Ad.5/3-mda-7 and BI-97C1 significantly inhibits the growth of human PC xenografts in nude mice and spontaneously induced PC in Hi-myc transgenic mice. Tumor growth inhibition correlated with increased TUNEL staining and decreased Ki-67 expression in both PC xenografts and prostates of Hi-myc mice. These findings demonstrate that pharmacological inhibition of Mcl-1 with the Apogossypol derivative, BI-97C1, sensitizes human PCs to mda-7/IL-24-mediated cytotoxicity, thus potentially augmenting the therapeutic benefit of this combinatorial approach toward PC.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Terapia Genética/métodos , Gossipol/análogos & derivados , Neoplasias da Próstata/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Gossipol/farmacologia , Gossipol/uso terapêutico , Humanos , Interleucinas/administração & dosagem , Masculino , Camundongos , Camundongos Nus , Proteína de Sequência 1 de Leucemia de Células Mieloides , Neoplasias da Próstata/patologia , Neoplasias da Próstata/terapia , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In melanoma, the activation of pro-survival signaling pathways, such as the AKT and NF-κB pathways, is critical for tumor growth. We have recently reported that the AKT inhibitor BI-69A11 causes efficient inhibition of melanoma growth. Here, we show that in addition to its AKT inhibitory activity, BI-69A11 also targets the NF-κB pathway. In melanoma cell lines, BI-69A11 inhibited TNF-α-stimulated IKKα/ß and IκB phosphorylation as well as NF-κB reporter gene expression. Furthermore, the effective inhibition of melanoma growth by BI-69A11 was attenuated upon NF-κB activation. Mechanistically, reduced NF-κB signaling by BI-69-A11 is mediated by the inhibition of sphingosine kinase 1, identified in a screen of 315 kinases. Significantly, we demonstrate that BI-69A11 is well tolerated and orally active against UACC 903 and SW1 melanoma xenografts. Our results demonstrate that BI-69A11 inhibits both the AKT and the NF-κB pathways and that the dual targeting of these pathways may be efficacious as a therapeutic strategy in melanoma.
Assuntos
Benzimidazóis/uso terapêutico , Melanoma/tratamento farmacológico , Terapia de Alvo Molecular , NF-kappa B/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Quinolonas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Neoplasias Cutâneas/tratamento farmacológico , Administração Oral , Animais , Benzimidazóis/administração & dosagem , Benzimidazóis/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Melanoma/enzimologia , Camundongos , Camundongos Nus , NF-kappa B/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinolonas/administração & dosagem , Quinolonas/farmacologia , Neoplasias Cutâneas/enzimologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We report comprehensive structure-activity relationship studies on a novel series of c-Jun N-terminal kinase (JNK) inhibitors. Intriguingly, the compounds have a dual inhibitory activity by functioning as both ATP and JIP mimetics, possibly by binding to both the ATP binding site and to the docking site of the kinase. Several of such novel compounds display potent JNK inhibitory profiles both in vitro and in cell.
Assuntos
Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/síntese química , Tiofenos/farmacologia , Trifosfato de Adenosina , Sítios de Ligação , Linhagem Celular , Desenho de Fármacos , Humanos , Mimetismo Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Relação Estrutura-Atividade , Tiofenos/químicaRESUMO
Our focus in the past several years has been on the identification of novel and effective pan-Bcl-2 antagonists. We have recently reported a series of Apogossypolone (ApoG2) derivatives, resulting in the chiral compound (±) BI97D6. We report here the synthesis and evaluation on its optically pure (-) and (+) atropisomers. Compound (-) BI97D6 potently inhibits the binding of BH3 peptides to Bcl-X(L), Bcl-2, Mcl-1, and Bfl-1 with IC(50) values of 76 ± 5, 31 ± 2, 25 ± 8, and 122 ± 28 nM, respectively. In a cellular assay, compound (-) BI97D6 effectively inhibits cell growth in the PC-3 human prostate cancer and H23 human lung cancer cell lines with EC(50) values of 0.22 ± 0.08 and 0.14 ± 0.02 µM, respectively. Similarly, compound (-) BI97D6 effectively induces apoptosis in the BP3 human lymphoma cell line in a dose-dependent manner. The compound also shows little cytotoxicity against bax(-/-)/bak(-/-) cells, suggesting that it kills cancers cells predominantly via a Bcl-2 pathway. Moreover, compound (-) BI97D6 displays in vivo efficacy in both a Bcl-2-transgenic mouse model and in a prostate cancer xenograft model in mice. Therefore, compound (-) BI97D6 represents a promising drug lead for the development of novel apoptosis-based therapies for cancer.
RESUMO
Overexpression of antiapoptotic Bcl-2 family proteins is commonly related with tumor maintenance, progression, and chemoresistance. Inhibition of these antiapoptotic proteins is an attractive approach for cancer therapy. Guided by nuclear magnetic resonance (NMR) binding assays, a series of 5,5' substituted compound 6a (Apogossypolone) derivatives was synthesized and identified pan-active antagonists of antiapoptotic Bcl-2 family proteins, with binding potency in the low micromolar to nanomolar range. Compound 6f inhibits the binding of BH3 peptides to Bcl-X(L), Bcl-2, and Mcl-1 with IC(50) values of 3.10, 3.12, and 2.05 µM, respectively. In a cellular assay, 6f potently inhibits cell growth in several human cancer cell lines in a dose-dependent manner. Compound 6f further displays in vivo efficacy in transgenic mice and demonstrated superior single-agent antitumor efficacy in a PPC-1 mouse xenograft model. Together with its negligible toxicity, compound 6f represents a promising drug lead for the development of novel apoptosis-based therapies for cancer.
Assuntos
Antineoplásicos/síntese química , Gossipol/análogos & derivados , Naftoquinonas/síntese química , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Estabilidade de Medicamentos , Feminino , Gossipol/síntese química , Gossipol/química , Gossipol/farmacologia , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Camundongos , Camundongos Nus , Camundongos Transgênicos , Microssomos/metabolismo , Modelos Moleculares , Naftoquinonas/química , Naftoquinonas/farmacologia , Transplante de Neoplasias , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Ratos , Relação Estrutura-Atividade , Transplante HeterólogoRESUMO
In our continued attempts to identify novel and effective pan-Bcl-2 antagonists, we have recently reported a series of compound 2 (Apogossypol) derivatives, resulting in the chiral compound 4 (8r). We report here the synthesis and evaluation on its optically pure individual isomers. Compound 11 (BI-97C1), the most potent diastereoisomer of compound 4, inhibits the binding of BH3 peptides to Bcl-X(L), Bcl-2, Mcl-1, and Bfl-1 with IC(50) values of 0.31, 0.32, 0.20, and 0.62 microM, respectively. The compound also potently inhibits cell growth of human prostate cancer, lung cancer, and lymphoma cell lines with EC(50) values of 0.13, 0.56, and 0.049 microM, respectively, and shows little cytotoxicity against bax(-/-)bak(-/-) cells. Compound 11 displays in vivo efficacy in transgenic mice models and also demonstrated superior single-agent antitumor efficacy in a prostate cancer mouse xenograft model. Therefore, compound 11 represents a potential drug lead for the development of novel apoptosis-based therapies against cancer.
Assuntos
Antineoplásicos/síntese química , Apoptose , Gossipol/análogos & derivados , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Ligação Competitiva , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Ensaios de Seleção de Medicamentos Antitumorais , Polarização de Fluorescência , Gossipol/síntese química , Gossipol/química , Gossipol/farmacologia , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Transplante de Neoplasias , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Estereoisomerismo , Transplante HeterólogoRESUMO
The E3 ubiquitin ligase Siah2 has been implicated in the regulation of the hypoxia response, as well as in the control of Ras, JNK/p38/NF-kappaB signaling pathways. Both Ras/mitogen-activated protein kinase (MAPK) and hypoxia pathways are important for melanoma development and progression, pointing to the possible use of Siah2 as target for treatment of this tumor type. In the present study, we have established a high-throughput electro-chemiluninescent-based assay in order to screen and identify inhibitors of Siah2 ubiquitin ligase activity. Of 1840 compounds screened, we identified and characterized menadione (MEN) as a specific inhibitor of Siah2 ligase activity. MEN attenuated Siah2 self-ubiquitination, and increased expression of its substrates PHD3 and Sprouty2, with concomitant decrease in levels of HIF-1alpha and pERK, the respective downstream effectors. MEN treatment no longer affected PHD3 or Sprouty2 in Siah-KO cells, pointing to its Siah-dependent effects. Further, MEN inhibition of Siah2 was not attenuated by free radical scavenger, suggesting it is ROS-independent. Significantly, growth of xenograft melanoma tumors was inhibited following the administration of MEN or its derivative. These findings reveal an efficient platform for the identification of Siah inhibitors while identifying and characterizing MEN as Siah inhibitor that attenuates hypoxia and MAPK signaling, and inhibits melanoma tumorigenesis.
Assuntos
Hipóxia/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Melanoma , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/metabolismo , Vitamina K 3/metabolismo , Animais , Bioensaio/métodos , Linhagem Celular , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Melanoma/metabolismo , Melanoma/patologia , Proteínas de Membrana , Camundongos , Camundongos Nus , Transplante de Neoplasias , Transplante Heterólogo , Ubiquitina-Proteína Ligases/genética , Vitamina K 3/química , Vitaminas/química , Vitaminas/metabolismoRESUMO
Guided by nuclear magnetic resonance (NMR) binding assays and computational docking studies, a series of 5,5' substituted apogossypol derivatives was synthesized that resulted in potent pan-active inhibitors of antiapoptotic Bcl-2 family proteins. Compound 8r inhibits the binding of BH3 peptides to Bcl-X(L), Bcl-2, Mcl-1, and Bfl-1 with IC(50) values of 0.76, 0.32, 0.28, and 0.73 microM, respectively. The compound also potently inhibits cell growth of human lung cancer and BP3 human B-cell lymphoma cell lines with EC(50) values of 0.33 and 0.66 microM, respectively. Compound 8r shows little cytotoxicity against bax(-/-)bak(-/-) cells, indicating that it kills cancers cells via the intended mechanism. The compound also displays in vivo efficacy in transgenic mice in which Bcl-2 is overexpressed in splenic B-cells. Together with its improved chemical, plasma, and microsomal stability relative to compound 2 (apogossypol), compound 8r represents a promising drug lead for the development of novel apoptosis-based therapies for cancer.
Assuntos
Apoptose/efeitos dos fármacos , Gossipol/análogos & derivados , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Alquilação , Amidas/química , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Gossipol/sangue , Gossipol/química , Gossipol/metabolismo , Gossipol/farmacologia , Humanos , Cetonas/química , Espectroscopia de Ressonância Magnética , Camundongos , Microssomos/metabolismo , Permeabilidade , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Especificidade por SubstratoRESUMO
Guided by a combination of nuclear magnetic resonance binding assays and computational docking studies, we synthesized a library of 5,5' substituted Apogossypol derivatives as potent Bcl-XL antagonists. Each compound was subsequently tested for its ability to inhibit Bcl-XL in an in vitro fluorescence polarization competition assay and exert single-agent proapoptotic activity in human cancer cell lines. The most potent compound BI79D10 binds to Bcl-XL, Bcl-2, and Mcl-1 with IC50 values of 190, 360, and 520 nmol/L, respectively, and potently inhibits cell growth in the H460 human lung cancer cell line with an EC50 value of 680 nmol/L, expressing high levels of Bcl-2. BI79D10 also effectively induces apoptosis of the RS11846 human lymphoma cell line in a dose-dependent manner and shows little cytotoxicity against bax-/-bak-/- mouse embryonic fibroblast cells, in which antiapoptotic Bcl-2 family proteins lack a cytoprotective phenotype, implying that BI79D10 has little off-target effects. BI79D10 displays in vivo efficacy in transgenic mice, in which Bcl-2 is overexpressed in splenic B cells. Together with its improved plasma and microsomal stability relative to Apogossypol, BI79D10 represents a lead compound for the development of novel apoptosis-based therapies for cancer.
Assuntos
Gossipol/análogos & derivados , Neoplasias Pulmonares/tratamento farmacológico , Linfoma/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Sobrevivência Celular/efeitos dos fármacos , Feminino , Polarização de Fluorescência , Gossipol/síntese química , Gossipol/química , Gossipol/farmacologia , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Linfoma/metabolismo , Linfoma/patologia , Espectroscopia de Ressonância Magnética , Masculino , Proteínas de Membrana/metabolismo , Membranas Artificiais , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Microssomos Hepáticos , Modelos Moleculares , Proteína de Sequência 1 de Leucemia de Células Mieloides , Fragmentos de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Ratos , Células Tumorais Cultivadas , Proteína Killer-Antagonista Homóloga a bcl-2/fisiologia , Proteína X Associada a bcl-2/fisiologia , Proteína bcl-X/metabolismoRESUMO
We report on a general structure- and NMR-based approach to derive druglike small molecule inhibitors of protein-protein interactions in a rapid and efficient manner. We demonstrate the utility of the approach by deriving novel and effective SMAC mimetics targeting the antiapoptotic protein X-linked inhibitor of apoptosis protein (XIAP). The XIAP baculovirus IAP repeat 3 (Bir3) domain binds directly to the N-terminal of caspase-9, thus inhibiting programmed cell death. It has been shown that in the cell this interaction can be displaced by the protein second mitochondrial activator of caspases (SMAC) and that its N-terminal tetrapeptide region (NH2-AVPI, Ala-Val-Pro-Ile) is responsible for this activity. However, because of their limited cell permeability, synthetic SMAC peptides are inefficient when tested in cultured cells, limiting their use as potential chemical tools or drug candidates against cancer cells. Hence, as an application, we report on the derivation of novel, selective, druglike, cell permeable SMAC mimics with cellular activity.