RESUMO
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 the synthesis and evaluation of an indazole-spin-labeled compound that was designed as an effective chemical probe for second site screening against the protein kinase JNK using NMR-based techniques. We demonstrate the utility of the derived compound in detecting and characterizing binding events at the protein kinase docking site. In addition, we report on the NMR-based design and synthesis of a bidentate compound spanning both the ATP site and the docking site. We show that the resulting compound has nanomolar affinity for JNK despite the relatively weak affinities of the individual fragments that constitute it. The approach demonstrates that targeting the docking site of protein kinases represents a valuable yet unexplored avenue to obtain potent kinase inhibitors with increased selectivity.
Assuntos
Óxidos N-Cíclicos/síntese química , Indazóis/síntese química , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Modelos Moleculares , Oligopeptídeos/síntese química , Marcadores de Spin/síntese química , Sítios de Ligação , Óxidos N-Cíclicos/química , Indazóis/química , Proteínas Quinases JNK Ativadas por Mitógeno/química , Espectroscopia de Ressonância Magnética , Oligopeptídeos/química , Ligação ProteicaRESUMO
Natural sweet proteins may be used as sugar replacer in simple liquid food systems but their applicability in more complex matrices has not been investigated yet. Gelling agent nature and texture characteristics as well as type and distribution of a stimulus in a gel could affect taste perception through inhibition or enhancement of tastants migration to the receptors. The mechanical, nonoral texture and time-intensity sweetness characteristics of sweet proteins MNEI and super sweet Y65R mutant, aspartame and saccharin added at a concentration iso-sweet to 40 g/L of sucrose in three agar gel concentrations (1%, 1.5%, and 2%) were evaluated. The results have shown that agar concentration and agar sweetener interaction particularly affect mechanical fracture stress and non oral hardness of the sweetened gels. Time intensity results illustrated that unlike in solution, the intensity of sweet taste in a gelled system over time decreases. Indeed, the behavior of the sweet proteins differed greatly in the gelled system compared to when they are in solution. PRACTICAL APPLICATIONS: MNEI has been proved to be a high-potency sweetener for beverages, but the possibility to use it in semisolid foodstuff was not investigated yet. This study represented a preliminary characterization of two variants of natural sweetener monellin, MNEI and Y65R in semisolid model foodstuff. The data were an important scientific contribution to the knowledge of sweet proteins in agar-based gels and could be useful in order to extend the possible application of these sweet proteins as low calorie sweeteners in semisolid foodstuffs. Some problems concerning their delivered sweetness in agar gels were underlined and their application should be optimized in order to improve sweetness conveyed.
Assuntos
Ágar/química , Géis/química , Proteínas de Plantas/química , Edulcorantes/química , Aspartame , Bebidas , Proteínas Mutantes/química , Sacarina , Sacarose , Percepção Gustatória , Fatores de TempoRESUMO
Overexpression of antiapoptotic members of the Bcl-2 family proteins, such as Bcl-x(L) and Mfl-1, has been shown to be involved in resistance to chemotherapeutic drugs in many forms of cancers. Recent efforts from the Abbott Laboratories resulted in the development of the acylsulfonamide compound and clinical candidate that targets selectively Bcl-2, Bcl-x(L), and Bcl-w while it is not active against Mcl-1 and Bfl-1. However, early clinical and preclinical studies suggest that pan-Bcl-2 antagonists, targeting simultaneously Mcl-1, Bcl-xL, and possibly all other four antiapoptotic Bcl-2 proteins, may result in more efficacious drugs. Here, following an NMR fragment-based approach, SAR by ILOEs, we report on compounds that exhibit nanomolar affinities for both Bcl-x(L) and Mcl-1 in vitro. We believe that these molecules can be used as useful starting point for the development of novel Bcl-2 antagonists, in particular targeting Mcl-1.
Assuntos
Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Sulfonamidas/química , Sulfonamidas/farmacologia , Proteína bcl-X/antagonistas & inibidores , Acilação , Humanos , Espectroscopia de Ressonância Magnética , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Proteína bcl-X/metabolismoRESUMO
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
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
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
Pin1 plays a key role in various biological cellular processes via the recognition of phosphorylated Ser/Thr-Proline motifs. Moreover, high expression levels of Pin1 are correlated to tumorgenesis in some cancer types. Here, we identify a novel series of small molecular weight compounds with a core structure mimicking the phoshorylated serine. The binding affinity and binding mode of the compounds for Pin1 are analyzed via NMR spectroscopy and computational studies. The reported chemical probes and relative binding data to Pin1 represent valuable stepping stones for the validation of Pin1 as target for drug discovery and for eventually the development of possible lead compounds.
Assuntos
Peptidilprolil Isomerase/antagonistas & inibidores , Peptidilprolil Isomerase/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Simulação por Computador , Escherichia coli/genética , Humanos , Ligantes , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Peptidilprolil Isomerase/química , Peptidilprolil Isomerase/genética , Ligação Proteica , Relação Quantitativa Estrutura-Atividade , Serina/química , Serina/metabolismoRESUMO
We report on the use of a diversity oriented synthesis (DOS) approach that resulted in the generation of a set of libraries of compounds presenting novel structural cores. These chemical cores have been employed to design potential antagonists of the antiapoptotic protein Bcl-x(L) through reiterated steps of molecular docking calculations followed by experimental verification of binding. Our data suggest that the DOS approach is suitable to generate novel scaffolds, which can be employed to target protein-protein interactions.
Assuntos
Descoberta de Drogas/métodos , Proteína bcl-X/antagonistas & inibidores , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína bcl-X/química , Proteína bcl-X/metabolismoRESUMO
Anti-apoptotic Bcl-2 family proteins such as Bcl-2 and Bcl-X(L) have been recently validated as targets for the discovery of novel anti-cancer agents. We previously reported that racemic (+/-) Apogossypol, a semi-synthetic compound derived from the natural product Gossypol, binds and inhibits Bcl-2 and Bcl-X(L)in vitro and in cell. Given that (+) and (-) Gossypol display different proapoptotic activities, here we report on the synthesis of (+) and (-) Apogossypol and the evaluation of their in vitro and cellular activity.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Gossipol/análogos & derivados , Proteína bcl-X/antagonistas & inibidores , Antineoplásicos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Polarização de Fluorescência , Gossipol/síntese química , Gossipol/farmacologia , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , EstereoisomerismoRESUMO
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
Successful cancer therapies aim to induce selective apoptosis in neoplastic cells. The current suboptimal efficiency and selectivity drugs have therapeutic limitations and induce concomitant side effects. Recently, novel cancer therapies based on the use of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) have emerged. TRAIL, a key component of the natural antitumor immune response, selectively kills many tumor cell types. Earlier studies with recombinant TRAIL, however, revealed its many shortcomings including a short half-life, off-target toxicity, and existence of TRAIL-resistant tumor cells. We improved the efficacy of recombinant TRAIL by redesigning its structure and the expression and purification procedures. The result is a highly stable leucine zipper (LZ)-TRAIL chimera that is simple to produce and purify. This chimera functions as a trimer in a manner that is similar to natural TRAIL. The formulation of the recombinant LZ-TRAIL we have developed has displayed high specific activity in both cell-based assays in vitro and animal tests in vivo. Our results have shown that the half-life of LZ-TRAIL is improved and now exceeds 1 h in mice compared with a half-life of only minutes reported earlier for recombinant TRAIL. We have concluded that our LZ-TRAIL construct will serve as a foundation for a new generation of fully human LZ-TRAIL proteins suitable for use in preclinical and clinical studies and for effective combination therapies to overcome tumor resistance to TRAIL.
Assuntos
Zíper de Leucina/genética , Neoplasias Mamárias Experimentais/tratamento farmacológico , Proteínas Recombinantes de Fusão/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/genética , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Citometria de Fluxo , Humanos , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Nus , Camundongos SCID , Neoplasias/metabolismo , Neoplasias/patologia , Engenharia de Proteínas , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Anti-apoptotic Bcl-2-family proteins (Bcl-2, Bcl-x(L), Bfl-1, Mcl-1, Bcl-W and Bcl-B) have been recently validated as drug discovery targets for cancer, owed to their ability to confer tumor resistance to chemotherapy or radiation. The anti-apoptotic activity of Bcl-2 proteins is due to their ability to heterodimerize with their pro-apoptotic counterparts (proteins such as Bad, Bim or Bid) via a conserved peptide region termed BH3. Thus, molecules that mimic pro-apoptotic BH3 domains represent a direct approach to overcoming the protective effects of anti-apoptotic proteins such as Bcl-2 and Bcl-x(L). Here, we report on the development and evaluation of two novel Lanthanide-based assays that are formatted for high-throughput screening of small molecules capable of antagonizing BH3-Bcl-2 interactions. The assay conditions, robustness and reproducibility (Z' factors) are described. These assays represent useful tools to enable further studies in the search for novel, safe and effective anti-cancer agents targeting Bcl-2-family proteins.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/antagonistas & inibidores , Elementos da Série dos Lantanídeos/química , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Sequência de Aminoácidos , Antineoplásicos/isolamento & purificação , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Transferência Ressonante de Energia de Fluorescência , Fluorimunoensaio , Humanos , Dados de Sequência Molecular , Reprodutibilidade dos TestesRESUMO
Apoptosis, or programmed cell death, plays a key role in normal tissue homeostasis ensuring a proper balance between cell production and cell loss. Anti-apoptotic Bcl-2-family proteins are central regulators of the apoptotic pathway and due to their ability to confer tumor resistance to chemotherapy or radiation, have been recently validated as targets for cancer drug discovery. Since the crucial interaction between pro- and anti-apoptotic members occurs via a conserved region located on the surface of the protein, a viable way to inhibit the anti-death activity of Bcl-2 proteins is to design small molecule inhibitors that occupy this cavity. Here, we describe a structure-based approach that led to the identification of four small molecule inhibitors directed at the hydrophobic groove on the surface of the Bcl-2 family protein Bcl-xL. The compounds were characterized in a number of assays including in vitro binding using 15N-labeled protein, a displacement DELFIA assay, and a cell-based viability assay with human cancer cells.
Assuntos
Antineoplásicos/química , Apoptose/efeitos dos fármacos , Proteína bcl-X/antagonistas & inibidores , Antineoplásicos/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sequência Conservada , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Imunoensaio , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Pathogens or their toxins, including influenza virus, Pseudomonas, and anthrax toxins, require processing by host proprotein convertases (PCs) to enter host cells and to cause disease. Conversely, inhibiting PCs is likely to protect host cells from multiple furin-dependent, but otherwise unrelated, pathogens. To determine if this concept is correct, we designed specific nanomolar inhibitors of PCs modeled from the extended cleavage motif TPQRERRRKKR downward arrowGL of the avian influenza H5N1 hemagglutinin. We then confirmed the efficacy of the inhibitory peptides in vitro against the fluorescent peptide, anthrax protective antigen (PA83), and influenza hemagglutinin substrates and also in mice in vivo against two unrelated toxins, anthrax and Pseudomonas exotoxin. Peptides with Phe/Tyr at P1' were more selective for furin. Peptides with P1' Thr were potent against multiple PCs. Our strategy of basing the peptide sequence on a furin cleavage motif known for an avian flu virus shows the power of starting inhibitor design with a known substrate. Our results confirm that inhibiting furin-like PCs protects the host from the distinct furin-dependent infections and lay a foundation for novel, host cell-focused therapies against acute diseases.
Assuntos
Furina/química , Pseudomonas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Antraz/metabolismo , Sítios de Ligação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Químicos , Dados de Sequência Molecular , Peptídeos/química , Ligação Proteica , Espectrometria de Fluorescência/métodosRESUMO
Bcl-2 family proteins play a crucial role in tissue homeostasis and apoptosis (programmed cell death). Bid is a proapoptotic member of the Bcl-2 family, promoting cell death when activated by caspase-8. Following an NMR-based approach (structure-activity relationships by interligand NOE) we were able to identify two chemical fragments that bind on the surface of Bid. Covalent linkage of the two fragments led to high-affinity bidentate derivatives. In vitro and in-cell assays demonstrate that the compounds prevent tBid translocation to the mitochondrial membrane and the subsequent release of proapoptotic stimuli and inhibit neuronal apoptosis in the low micromolar range. Therefore, by using a rational chemical-biology approach, we derived antiapoptotic compounds that may have a therapeutic potential for disorders associated with Bid activation, e.g., neurodegenerative diseases, cerebral ischemia, or brain trauma.