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1.
Chem Res Toxicol ; 33(1): 154-161, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31461269

RESUMO

Despite the recent advances in the life sciences and the remarkable investment in drug discovery research, the success rate of small-molecule drug development remains low. Safety is the second most influential factor of drug attrition in clinical studies; thus, the selection of compounds with fewer toxicity concerns is crucial to increase the success rate of drug discovery. Compounds that promiscuously bind to multiple targets are likely to cause unexpected pharmacological activity that may lead to adverse effects. Therefore, avoiding such compounds during early research stages would contribute to identifying compounds with a higher chance of success in the clinic. To evaluate the interaction profile against a wide variety of targets, we constructed a small-scale promiscuity panel (PP) consisting of eight targets (ROCK1, PDE4D2, GR, PPARγ, 5-HT2B, adenosine A3, M1, and GABAA) that were selected from diverse gene families. The validity of this panel was confirmed by comparison with the promiscuity index evaluated from larger-scale panels. Analysis of data from the PP revealed that both lipophilicity and basicity are likely to increase promiscuity, while the molecular weight does not significantly contribute. Additionally, the promiscuity assessed using our PP correlated with the occurrence of both in vitro cytotoxicity and in vivo toxicity, suggesting that the PP is useful to identify compounds with fewer toxicity concerns. In summary, this small-scale and cost-effective PP can contribute to the identification of safer compounds that would lead to a reduction in drug attrition due to safety issues.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Animais , Sobrevivência Celular , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/prevenção & controle , Células Hep G2 , Humanos , Camundongos , PPAR gama/genética , Ratos , Receptor A3 de Adenosina/genética , Receptor Muscarínico M1/genética , Receptor 5-HT2B de Serotonina/genética , Receptores de GABA-A/genética , Receptores de Glucocorticoides/genética , Quinases Associadas a rho/genética
2.
Pharmacol Res Perspect ; 7(5): e00517, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31508234

RESUMO

Enteropeptidase, localized into the duodenum brush border, is a key enzyme catalyzing the conversion of pancreatic trypsinogen proenzyme to active trypsin, thereby regulating protein digestion and energy homeostasis. We report the discovery and pharmacological profiles of SCO-792, a novel inhibitor of enteropeptidase. A screen employing fluorescence resonance energy transfer was performed to identify enteropeptidase inhibitors. Inhibitory profiles were determined by in vitro assays. To evaluate the in vivo inhibitory effect on protein digestion, an oral protein challenge test was performed in rats. Our screen identified a series of enteropeptidase inhibitors, and compound optimization resulted in identification of SCO-792, which inhibited enteropeptidase activity in vitro, with IC 50 values of 4.6 and 5.4 nmol/L in rats and humans, respectively. In vitro inhibition of enteropeptidase by SCO-792 was potentiated by increased incubation time, and the calculated Kinact/KI was 82 000/mol/L s. An in vitro dissociation assay showed that SCO-792 had a dissociation half-life of almost 14 hour, with a calculated koff rate of 0.047/hour, which suggested that SCO-792 is a reversible enteropeptidase inhibitor. In normal rats, a ≤4 hour prior oral dose of SCO-792 effectively inhibited plasma elevation of branched-chain amino acids in an oral protein challenge test, which indicated that SCO-792 effectively inhibited protein digestion in vivo. In conclusion, our new screen system identified SCO-792 as a potent and reversible inhibitor against enteropeptidase. SCO-792 slowly dissociated from enteropeptidase in vitro and inhibited protein digestion in vivo. Further study using SCO-792 could reveal the effects of inhibiting enteropeptidase on biological actions.


Assuntos
Enteropeptidase/antagonistas & inibidores , Inibidores Enzimáticos/administração & dosagem , Bibliotecas de Moléculas Pequenas/administração & dosagem , Administração Oral , Animais , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Transferência Ressonante de Energia de Fluorescência , Humanos , Concentração Inibidora 50 , Ratos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
3.
Bioorg Med Chem Lett ; 29(21): 126641, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31526603

RESUMO

Selectivity profiling of compounds is important for kinase drug discovery. To this end, we aimed to develop a broad-range protein kinase assay by synthesizing a novel staurosporine-derived fluorescent probe based on staurosporine and kinase-binding related structural information. Upon structural analysis of staurosporine with kinases, a 4'-methylamine moiety of staurosporine was found to be located on the solvent side of the kinases, to which several linker units can be conjugated by either alkylation or acylation. However, such conjugation was suggested to reduce the binding affinities of the modified compound for several kinases, owing to the elimination of hydrogen bond donor moiety of NH-group from 4'-methylamine and/or steric hindrance by acyl moiety. Based on this structural information, we designed and synthesized a novel staurosporine-based probe without methyl group in order to retain the hydrogen bond donor, similar to unmodified staurosporine. The broad range of the kinase binding assay demonstrated that our novel fluorescent probe is an excellent tool for developing broad-ranging kinase binding assay.


Assuntos
Corantes Fluorescentes/química , Inibidores de Proteínas Quinases/química , Proteínas Quinases/química , Estaurosporina/química , Sítios de Ligação , Ligação Competitiva , Técnicas Biossensoriais , Avaliação Pré-Clínica de Medicamentos/métodos , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/síntese química , Humanos , Ligação de Hidrogênio , Metilaminas/química , Estrutura Molecular , Ligação Proteica , Sensibilidade e Especificidade , Estaurosporina/síntese química , Relação Estrutura-Atividade
4.
Biochemistry ; 57(8): 1369-1379, 2018 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-29293322

RESUMO

B-cell lymphoma 6 (BCL6) is the most frequently involved oncogene in diffuse large B-cell lymphomas (DLBCLs). BCL6 shows potent transcriptional repressor activity through interactions with its corepressors, such as BCL6 corepressor (BCOR). The inhibition of the protein-protein interaction (PPI) between BCL6 and its corepressors suppresses the growth of BCL6-dependent DLBCLs, thus making BCL6 an attractive drug target for lymphoma treatment. However, potent small-molecule PPI inhibitor identification remains challenging because of the lack of deep cavities at PPI interfaces. This article reports the discovery of a potent, cell-active small-molecule BCL6 inhibitor, BCL6-i (8), that operates through irreversible inhibition. First, we synthesized irreversible lead compound 4, which targets Cys53 in a cavity on the BCL6-BTB domain dimer by introducing an irreversible warhead to high-throughput screening hit compound 1. Further chemical optimization of 4 based on kinact/KI evaluation produced BCL6-i with a kinact/KI value of 1.9 × 104 M-1 s-1, corresponding to a 670-fold improvement in potency compared to that of 4. By exploiting the property of irreversible inhibition, engagement of BCL6-i to intracellular BCL6 was confirmed. BCL6-i showed intracellular PPI inhibitory activity between BCL6 and its corepressors, thus resulting in BCL6-dependent DLBCL cell growth inhibition. BCL6-i is a cell-active chemical probe with the most potent BCL6 inhibitory activity reported to date. The discovery process of BCL6-i illustrates the utility of irreversible inhibition for identifying potent chemical probes for intractable target proteins.


Assuntos
Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-6/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Linhagem Celular Tumoral , Cisteína/análise , Cisteína/metabolismo , Descoberta de Drogas , Humanos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/metabolismo , Modelos Moleculares , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-6/química , Bibliotecas de Moléculas Pequenas/química
5.
J Med Chem ; 60(21): 8963-8981, 2017 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-29023121

RESUMO

The discovery and optimization of Δ-5 desaturase (D5D) inhibitors are described. Investigation of the 1,3-oxazolidin-2-one scaffold was inspired by a pharmacophore model constructed from the common features of several hit compounds, resulting in the identification of 3,5-diphenyl-1,3-oxazolidin-2-one 5h as a novel lead showing potent in vitro activity. Subsequent optimization focused on the modification of two metabolic sites, which provided (4S,5S)-5i, a derivative with improved metabolic stability. Moreover, adding a substituent into the upper phenyl moiety further enhanced the intrinsic activity, which led to the discovery of 5-[(4S,5S)-5-(4fluorophenyl)-4-methyl-2-oxo-1,3-oxazolidin-3-yl]benzene-1,3-dicarbonitrile (4S,5S)-5n, endowed with excellent D5D binding affinity, cellular activity, and high oral bioavailability in a mouse. It exhibited robust in vivo hepatic arachidonic acid/dihomo-γ-linolenic acid ratio reduction (a target engagement marker) in an atherosclerosis mouse model. Finally, an asymmetric synthetic procedure for this compound was established.


Assuntos
Ácidos Graxos Dessaturases/antagonistas & inibidores , Oxazolidinonas/farmacologia , Administração Oral , Animais , Ácido Araquidônico/metabolismo , Aterosclerose/tratamento farmacológico , Disponibilidade Biológica , Dessaturase de Ácido Graxo Delta-5 , Descoberta de Drogas/métodos , Fígado/metabolismo , Camundongos , Oxazolidinonas/síntese química , Oxazolidinonas/metabolismo , Oxazolidinonas/farmacocinética , Relação Estrutura-Atividade
6.
Bioorg Med Chem ; 25(14): 3768-3779, 2017 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-28571972

RESUMO

A lead compound A was identified previously as an stearoyl coenzyme A desaturase (SCD) inhibitor during research on potential treatments for obesity. This compound showed high SCD1 binding affinity, but a poor pharmacokinetic (PK) profile and limited chemical accessibility, making it suboptimal for use in anticancer research. To identify potent SCD1 inhibitors with more promising PK profiles, we newly designed a series of 'non-spiro' 4, 4-disubstituted piperidine derivatives based on molecular modeling studies. As a result, we discovered compound 1a, which retained moderate SCD1 binding affinity. Optimization around 1a was accelerated by analyzing Hansch-Fujita and Hammett constants to obtain 4-phenyl-4-(trifluoromethyl)piperidine derivative 1n. Fine-tuning of the azole moiety of 1n led to compound 1o (T-3764518), which retained nanomolar affinity and exhibited an excellent PK profile. Reflecting the good potency and PK profile, orally administrated compound 1o showed significant pharmacodynamic (PD) marker reduction (at 0.3mg/kg, bid) in HCT116 mouse xenograft model and tumor growth suppression (at 1mg/kg, bid) in 786-O mouse xenograft model. In conclusion, we identified a new series of SCD1 inhibitors, represented by compound 1o, which represents a promising new chemical tool suitable for the study of SCD1 biology as well as the potential development of novel anticancer therapies.


Assuntos
Antineoplásicos/química , Inibidores Enzimáticos/síntese química , Oxidiazóis/síntese química , Piridazinas/síntese química , Estearoil-CoA Dessaturase/antagonistas & inibidores , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Células HCT116 , Humanos , Concentração Inibidora 50 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microssomos Hepáticos/metabolismo , Oxidiazóis/farmacocinética , Oxidiazóis/uso terapêutico , Oxidiazóis/toxicidade , Piperidinas/química , Piperidinas/metabolismo , Piperidinas/farmacologia , Ligação Proteica , Piridazinas/farmacocinética , Piridazinas/uso terapêutico , Piridazinas/toxicidade , Compostos de Espiro/química , Estearoil-CoA Dessaturase/metabolismo , Relação Estrutura-Atividade , Transplante Heterólogo
7.
Biochemistry ; 56(23): 2921-2927, 2017 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-28520393

RESUMO

Recently, there have been a limited number of new, validated targets for small-molecule drug discovery in the pharmaceutical industry. Although there are approximately 30 000 genes in the human genome, only 2% are targeted by currently approved small-molecule drugs. One reason that many targets remain neglected by drug discovery programs is the absence of biochemical assays enabling evaluation of the potency of inhibitors in a quantitative and high-throughput manner. To overcome this issue, we developed a biochemical assay to evaluate the potency of both reversible and irreversible inhibitors using a nonspecific thiol-labeling fluorescent probe. The assay can be applied to any targets with a cysteine residue in a pocket that can accommodate small-molecule ligands. By constructing a mathematical model, we showed that the potency of compounds can be quantitatively evaluated by performing an activity-based protein profiling assay. In addition, the validity of the theory was confirmed experimentally using epidermal growth factor receptor kinase as a model target. This approach provides an assay system for targets for which biochemical assays cannot be developed. Our approach can potentially not only expand the number of exploitable targets but also accelerate the lead optimization process by providing quantitative structure-activity relationship information.


Assuntos
Compostos de Boro/metabolismo , Descoberta de Drogas/métodos , Receptores ErbB/antagonistas & inibidores , Corantes Fluorescentes/metabolismo , Maleimidas/metabolismo , Modelos Moleculares , Inibidores de Proteínas Quinases/farmacologia , Reagentes de Sulfidrila/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Biocatálise , Compostos de Boro/química , Domínio Catalítico , Cisteína/química , Receptores ErbB/química , Receptores ErbB/genética , Receptores ErbB/metabolismo , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Ensaios de Triagem em Larga Escala , Humanos , Cinética , Ligantes , Maleimidas/química , Conformação Molecular , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Relação Quantitativa Estrutura-Atividade , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Células Sf9 , Spodoptera , Reagentes de Sulfidrila/química
8.
Eur J Pharmacol ; 807: 21-31, 2017 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-28442322

RESUMO

Most cancer cells are characterized by elevated lipid biosynthesis. The rapid proliferation of cancer cells requires de novo synthesis of fatty acids. Stearoyl-CoA desaturase-1 (SCD1), a key enzyme for lipogenesis, is overexpressed in various types of cancer and plays an important role in cancer cell proliferation. Therefore, it has been studied as a candidate target for cancer therapy. In this study, we demonstrate the pharmacological properties of T-3764518, a novel and orally available small molecule inhibitor of SCD1. T-3764518 inhibited stearoyl-CoA desaturase-catalyzed conversion of stearoyl-CoA to oleoyl-CoA in colorectal cancer HCT-116 cells and their growth. Further, it slowed tumor growth in an HCT-116 and a mesothelioma MSTO-211H mouse xenograft model. Comprehensive lipidomic analyses revealed that T-3764518 increases the membrane ratio of saturated: unsaturated fatty acids in various lipid species such as phosphatidylcholines and diacylglycerols in both cultured cells and HCT-116 xenografts. Treatment-associated lipidomic changes were followed by activated endoplasmic reticulum (ER) stress responses such as increased immunoglobulin heavy chain-binding protein expression in HCT-116 cells. These T-3764518-induced changes led to an increase in cleaved poly (ADP-ribose) polymerase 1 (PARP1), a marker of apoptosis. Additionally, bovine serum albumin conjugated with oleic acid, an SCD1 product, prevented cell growth inhibition and ER stress responses by T-3764518, indicating that these outcomes were not attributable to off-target effects. These results indicate that T-3764518 is a promising new anticancer drug candidate.


Assuntos
Antineoplásicos/farmacologia , Antineoplásicos/farmacocinética , Inibidores Enzimáticos/farmacologia , Oxidiazóis/farmacologia , Oxidiazóis/farmacocinética , Piridazinas/farmacologia , Piridazinas/farmacocinética , Estearoil-CoA Dessaturase/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/metabolismo , Apoptose/efeitos dos fármacos , Disponibilidade Biológica , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacocinética , Ácidos Graxos/metabolismo , Células HCT116 , Humanos , Camundongos , Oxidiazóis/administração & dosagem , Oxidiazóis/metabolismo , Piridazinas/administração & dosagem , Piridazinas/metabolismo , Estearoil-CoA Dessaturase/metabolismo
9.
SLAS Discov ; 22(9): 1168-1174, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28426937

RESUMO

In a high-throughput screening (HTS) process, the chemical reactivity of test samples should be carefully examined because such reactive compounds may lead to false-positive results and adverse effects in vivo. Among all natural amino acids, the thiol side chain in cysteine has the highest nucleophilicity; thus, assessment of intrinsic thiol group reactivity in the HTS processes is expected to accelerate drug discovery. In general, kchem (M-1s-1), the secondary reaction rate constant of a compound to thiol, can be evaluated via time course measurements of thiol-compound adducts using liquid chromatography-mass spectroscopy; this requires time-consuming and labor-intensive procedures. To overcome this issue, we developed a fluorescence-based competitive endpoint assay that allows quantitative calculation of the reaction rate of test compounds in an HTS format. Our assay is based on the competitive reaction for a free thiol (e.g., glutathione) between the test compounds and a fluorescent probe, o-maleimide BODIPY. Our assay provides robust data with a satisfactory throughput at an affordable cost. Our kchem evaluation method has advantages over previous assays in terms of higher throughput and quantitativeness. Thus, it contributes to early elimination of reactive compounds as well as quantitative evaluation of the kchem values of covalent inhibitors.

10.
PLoS One ; 11(11): e0166198, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27832159

RESUMO

Obesity is now recognized as a state of chronic low-grade inflammation and is called as metabolic inflammation. Delta-5 desaturase (D5D) is an enzyme that metabolizes dihomo-γ-linolenic acid (DGLA) to arachidonic acid (AA). Thus, D5D inhibition increases DGLA (precursor to anti-inflammatory eicosanoids) while decreasing AA (precursor to pro-inflammatory eicosanoids), and could result in synergistic improvement in the low-grade inflammatory state. Here, we demonstrate reduced insulin resistance and the anti-obesity effect of a D5D selective inhibitor (compound-326), an orally active small-molecule, in a high-fat diet-induced obese (DIO) mouse model. In vivo D5D inhibition was confirmed by determining changes in blood AA/DGLA profiles. In DIO mice, chronic treatment with compound-326 lowered insulin resistance and caused body weight loss without significant impact on cumulative calorie intake. Decreased macrophage infiltration into adipose tissue was expected from mRNA analysis. Increased daily energy expenditure was also observed following administration of compound-326, in line with sustained body weight loss. These data indicate that the novel D5D selective inhibitor, compound-326, will be a new class of drug for the treatment of obese and diabetic patients.


Assuntos
Peso Corporal/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Ácidos Graxos Dessaturases/antagonistas & inibidores , Resistência à Insulina , Obesidade/prevenção & controle , Pirimidinonas/farmacologia , Pirrolidinonas/farmacologia , Ácido 8,11,14-Eicosatrienoico/sangue , Ácido 8,11,14-Eicosatrienoico/metabolismo , Adiponectina/genética , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Ácido Araquidônico/sangue , Ácido Araquidônico/metabolismo , Dessaturase de Ácido Graxo Delta-5 , Dieta Hiperlipídica/efeitos adversos , Metabolismo Energético/efeitos dos fármacos , Ácidos Graxos Dessaturases/metabolismo , Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/prevenção & controle , Leptina/genética , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Obesidade/etiologia , Obesidade/fisiopatologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Redução de Peso/efeitos dos fármacos
11.
ACS Med Chem Lett ; 7(9): 868-72, 2016 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-27660693

RESUMO

Delta-5 desaturase (D5D) catalyzes the conversion from dihomo-gamma linoleic acid (DGLA) to arachidonic acid (AA). DGLA and AA are common precursors of anti- and pro-inflammatory eicosanoids, respectively, making D5D an attractive drug target for inflammatory-related diseases. Despite several reports on D5D inhibitors, their biochemical mechanisms of action (MOAs) remain poorly understood, primarily due to the difficulty in performing quantitative enzymatic analysis. Herein, we report a radioligand binding assay to overcome this challenge and characterized T-3364366, a thienopyrimidinone D5D inhibitor, by use of the assay. T-3364366 is a reversible, slow-binding inhibitor with a dissociation half-life in excess of 2.0 h. The long residence time was confirmed in cellular washout assays. Domain swapping experiments between D5D and D6D support [(3)H]T-3364366 binding to the desaturase domain of D5D. The present study is the first to demonstrate biochemical MOA of desaturase inhibitors, providing important insight into drug discovery of desaturase enzymes.

12.
Angew Chem Int Ed Engl ; 54(47): 14099-102, 2015 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-26426864

RESUMO

Owing to their covalent target occupancy, irreversible inhibitors require low exposures and offer long duration, and their use thus represents a powerful strategy for achieving pharmacological efficacy. Importantly, the potency metric of irreversible inhibitors is kinact/KI not IC50. A simple approach to measuring kinact/KI was developed that makes use of an irreversible probe for competitive assays run to completion against test compounds. In this system, the kinact/KI value of the test compound is equal to (kinact/KI)probe ×[probe]/IC50. The advantages of this method include simplicity, high throughput, and application to all target classes, and it only requires an in-depth kinetic evaluation of the probe.


Assuntos
Determinação de Ponto Final , Inibidores Enzimáticos/farmacologia , Receptores ErbB/antagonistas & inibidores , Morfolinas/farmacologia , Inibidores Enzimáticos/química , Receptores ErbB/metabolismo , Humanos , Cinética , Estrutura Molecular , Morfolinas/química , Relação Estrutura-Atividade , Fatores de Tempo
13.
Bioorg Med Chem Lett ; 24(24): 5836-5839, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-25452002

RESUMO

Identification of inhibitors for protein-protein interactions (PPIs) from high-throughput screening (HTS) is challenging due to the weak affinity of primary hits. We present a hit validation strategy of PPI inhibitors using quantitative ligand displacement assay. From an HTS for Bcl-xL/Mcl-1 inhibitors, we obtained a hit candidate, I1, which potentially forms a reactive Michael acceptor, I2, inhibiting Bcl-xL/Mcl-1 through covalent modification. We confirmed rapid reversible and competitive binding of I1 with a probe peptide, suggesting non-covalent binding. The advantages of our approach over biophysical assays include; simplicity, higher throughput, low protein consumption and universal application to PPIs including insoluble membrane proteins.


Assuntos
Cetoácidos/química , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteína bcl-X/metabolismo , Ligação Competitiva , Butiratos/química , Butiratos/metabolismo , Ensaios de Triagem em Larga Escala , Cetoácidos/metabolismo , Cinética , Ligantes , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Domínios e Motivos de Interação entre Proteínas , Proteína bcl-X/antagonistas & inibidores
14.
J Med Chem ; 56(23): 9635-45, 2013 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-24215352

RESUMO

Mcl-1 and Bcl-xL are crucial regulators of apoptosis, therefore dual inhibitors of both proteins could serve as promising new anticancer drugs. To design Mcl-1/Bcl-xL dual inhibitors, we performed structure-guided analyses of the corresponding selective Mcl-1 and Bcl-xL inhibitors. A cocrystal structure of a pyrazolo[1,5-a]pyridine derivative with Mcl-1 protein was successfully determined and revealed the protein-ligand binding mode. The key structure for Bcl-xL inhibition was further confirmed through the substructural analysis of ABT-263, a representative Bcl-xL/Bcl-2/Bcl-w inhibitor developed by Abbott Laboratories. On the basis of the structural data from this analysis, we designed hybrid compounds by tethering the Mcl-1 and Bcl-xL inhibitors together. The results of X-ray crystallographic analysis of hybrid compound 10 in complexes with both Mcl-1 and Bcl-xL demonstrated its binding mode with each protein. Following further optimization, compound 11 showed potent Mcl-1/Bcl-xL dual inhibitory activity (Mcl-1, IC50 = 0.088 µM; and Bcl-xL, IC50 = 0.0037 µM).


Assuntos
Apoptose/efeitos dos fármacos , Compostos de Bifenilo/síntese química , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Sulfonamidas/síntese química , Proteína bcl-X/antagonistas & inibidores , Compostos de Anilina/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Compostos de Bifenilo/química , Compostos de Bifenilo/farmacologia , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Modelos Moleculares , Pirazóis/síntese química , Pirazóis/farmacologia , Piridinas/síntese química , Piridinas/farmacologia , Sulfonamidas/química , Sulfonamidas/farmacologia
15.
Chem Biol ; 14(6): 613-21, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17584609

RESUMO

Flavonoids and stilbenes have attracted much attention as potential targets for nutraceuticals, cosmetics, and pharmaceuticals. We have developed a system for producing "unnatural" flavonoids and stilbenes in Escherichia coli. The artificial biosynthetic pathway included three steps. These included a substrate synthesis step for CoA esters synthesis from carboxylic acids by 4-coumarate:CoA ligase, a polyketide synthesis step for conversion of the CoA esters into flavanones by chalcone synthase and chalcone isomerase, and into stilbenes by stilbene synthase, and a modification step for modification of the flavanones by flavone synthase, flavanone 3beta-hydroxylase and flavonol synthase. Incubation of the recombinant E. coli with exogenously supplied carboxylic acids led to production of 87 polyketides, including 36 unnatural flavonoids and stilbenes. This system is promising for construction of a larger library by employing other polyketide synthases and modification enzymes.


Assuntos
Escherichia coli , Flavonoides , Plantas , Estilbenos , Ácidos Carboxílicos/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Flavonoides/biossíntese , Flavonoides/química , Flavonoides/genética , Genes de Plantas/fisiologia , Estrutura Molecular , Plantas/enzimologia , Plantas/genética , Plantas/metabolismo , Plasmídeos , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Estilbenos/química , Especificidade por Substrato
16.
Appl Microbiol Biotechnol ; 73(5): 1143-9, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16960736

RESUMO

For production of genistein from N-acetylcysteamine-attached p-coumarate (p-coumaroyl-NAC) supplemented to the medium, a chalcone synthase (CHS) gene from Glycyrrhiza echinata, a chalcone isomerase (CHI) gene from Pueraria lobata, and an isoflavone synthase (IFS) gene from G. echinata were placed under the control of the galactose-inducible GAL promoters in pESC vector and were introduced in Saccharomyces cerevisiae. When the recombinant yeast cells (0.5 g wet weight) were used as "enzyme bags" and incubated at 30 degrees C for 48 h in 100 ml of the buffer containing galactose and 1 mM (265 mg/l) p-coumaroyl-NAC, ca. 340 microg genistein/l was produced. Another system consisting of two enzyme bags was also generated for the purpose of production of genistein from tyrosine. One enzyme bag was an Escherichia coli cell containing a phenylalanine ammonia-lyase gene from a yeast, a 4-coumarate/cinnamate:CoA ligase gene from the actinomycete Streptomyces coelicolor A3(2), the CHS gene, and the CHI gene, in addition to the acetyl-CoA carboxylase gene from Corynebacterium glutamicum, all of which were under the control of the isopropyl-beta-D-thiogalactopyranoside-inducible T7 promoter, and thus producing (S)-naringenin from tyrosine. The other enzyme bag was a S. cerevisiae cell containing the IFS gene. Coincubation of the E. coli cells (0.5 g wet weight) and S. cerevisiae cells (0.5 g wet weight) at 26 degrees C for 60 h in 20 ml of the buffer containing 3 mM (543 mg/l) tyrosine as the starting substrate yielded ca. 6 mg genistein/l.


Assuntos
Escherichia coli/metabolismo , Genisteína/metabolismo , Saccharomyces cerevisiae/metabolismo , Tirosina/metabolismo , Acetil-CoA Carboxilase/genética , Aciltransferases/genética , Clonagem Molecular , Coenzima A Ligases/genética , Corynebacterium glutamicum/enzimologia , Corynebacterium glutamicum/genética , Ácidos Cumáricos/metabolismo , Escherichia coli/genética , Flavanonas/biossíntese , Expressão Gênica , Glycyrrhiza/enzimologia , Glycyrrhiza/genética , Liases Intramoleculares/genética , Isopropiltiogalactosídeo/análogos & derivados , Isopropiltiogalactosídeo/farmacologia , Oxigenases/genética , Fenilalanina Amônia-Liase/genética , Regiões Promotoras Genéticas , Propionatos , Pueraria/enzimologia , Pueraria/genética , Saccharomyces cerevisiae/genética , Streptomyces coelicolor/enzimologia , Streptomyces coelicolor/genética , Temperatura , Fatores de Tempo
17.
Appl Microbiol Biotechnol ; 71(1): 53-8, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16133333

RESUMO

(2S)-Flavanones (naringenin and pinocembrin) are key intermediates in the flavonoid biosynthetic pathway in plants. Recombinant Escherichia coli cells containing four genes for a phenylalanine ammonia-lyase, cinnamate/coumarate:CoA ligase, chalcone synthase, and chalcone isomerase, in addition to the acetyl-CoA carboxylase, have been established for efficient production of (2S)-naringenin from tyrosine and (2S)-pinocembrin from phenylalanine. Further introduction of the flavone synthase I gene from Petroselinum crispum under the control of the T7 promoter and the synthetic ribosome-binding sequence in pACYCDuet-1 caused the E. coli cells to produce flavones: apigenin (13 mg/l) from tyrosine and chrysin (9.4 mg/l) from phenylalanine. Introduction into the E. coli cells of the flavanone 3beta-hydroxylase and flavonol synthase genes from the plant Citrus species led to production of flavonols: kaempferol (15.1 mg/l) from tyrosine and galangin (1.1 mg/l) from phenylalanine. The combinatorial biosynthesis of the flavones and flavonols in E. coli is promising for the construction of a library of various flavonoid compounds and un-natural flavonoids in bacteria.


Assuntos
Escherichia coli/metabolismo , Flavonas/biossíntese , Flavonóis/biossíntese , Engenharia de Proteínas , Citrus/enzimologia , Citrus/genética , Escherichia coli/genética , Flavonas/química , Flavonóis/química , Oxigenases de Função Mista/genética , Oxirredutases/genética , Petroselinum/enzimologia , Petroselinum/genética , Proteínas de Plantas/genética
18.
Appl Microbiol Biotechnol ; 68(4): 498-504, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15770480

RESUMO

For the fermentative production of plant-specific flavanones (naringenin, pinocembrin) by Escherichia coli, a plasmid was constructed which carried an artificial biosynthetic gene cluster, including PAL encoding a phenylalanine ammonia-lyase from a yeast, ScCCL encoding a cinnamate/coumarate:CoA ligase from the actinomycete Streptomyces coelicolor A3(2), CHS encoding a chalcone synthase from a licorice plant and CHI encoding a chalcone isomerase from the Pueraria plant. The recombinant E. coli cells produced (2S)-naringenin from tyrosine and (2S)-pinocembrin from phenylalanine. When the two subunit genes of acetyl-CoA carboxylase from Corynebacterium glutamicum were expressed under the control of the T7 promoter and the ribosome-binding sequence in the recombinant E. coli cells, the flavanone yields were greatly increased, probably because enhanced expression of acetyl-CoA carboxylase increased a pool of malonyl-CoA that was available for flavanone synthesis. Under cultural conditions where E. coli at a cell density of 50 g/l was incubated in the presence of 3 mM tyrosine or phenylalanine, the yields of naringenin and pinocembrin reached about 60 mg/l. The fermentative production of flavanones in E. coli is the first step in the construction of a library of flavonoid compounds and un-natural flavonoids in bacteria.


Assuntos
Flavanonas/biossíntese , Genes de Plantas/fisiologia , Genes Sintéticos , Engenharia Genética , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Flavanonas/genética , Malonil Coenzima A/metabolismo
19.
J Bacteriol ; 186(7): 2206-11, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15028707

RESUMO

In the model of the A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) regulatory cascade in Streptomyces griseus, A-factor binds ArpA, the A-factor receptor protein, that has bound to the adpA promoter and dissociates it from the DNA, thus inducing the transcription of adpA. AdpA switches on the transcription of a number of genes required for secondary metabolism and morphological differentiation, forming an AdpA regulon. Consistent with this model, arpA null mutants produced streptomycin and a yellow pigment in larger amounts and formed aerial hyphae from an earlier growth stage than the wild-type strain. On the other hand, mutant MK2, expressing a mutant ArpA (Trp119Ala), neither produced secondary metabolites nor formed aerial hyphae, because this A-factor-insensitive mutant ArpA always bound to and repressed the adpA promoter due to the amino acid replacement of Trp-119 with Ala. Introduction of adpA under the control of a foreign promoter into mutant MK2 restored all of the phenotypes that we could observe, which suggests that the only significant target of ArpA is adpA. In contrast to other gamma-butyrolactone regulatory systems, disruption of arpA had no effect on A-factor production, indicating that ArpA does not regulate A-factor biosynthesis. Instead, A-factor production was found to be repressed by AdpA in a two-step regulatory feedback loop.


Assuntos
4-Butirolactona/análogos & derivados , 4-Butirolactona/biossíntese , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas Repressoras/metabolismo , Streptomyces griseus/metabolismo , Transativadores/metabolismo , 4-Butirolactona/metabolismo , Proteínas de Bactérias/genética , Meios de Cultura , Proteínas de Ligação a DNA/genética , Mutação , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Streptomyces griseus/crescimento & desenvolvimento , Estreptomicina/metabolismo , Transativadores/genética , Transcrição Gênica
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