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/genéticaRESUMO
General control nonderepressible 2 (GCN2) is a master regulator kinase of amino acid homeostasis and important for cancer survival in the tumor microenvironment under amino acid depletion. We initiated studies aiming at the discovery of novel GCN2 inhibitors as first-in-class antitumor agents and conducted modification of the substructure of sulfonamide derivatives with expected type I half binding on GCN2. Our synthetic strategy mainly corresponding to the αC-helix allosteric pocket of GCN2 led to significant enhancement in potency and a good pharmacokinetic profile in mice. In addition, compound 6d, which showed slow dissociation in binding on GCN2, demonstrated antiproliferative activity in combination with the asparagine-depleting agent asparaginase in an acute lymphoblastic leukemia (ALL) cell line, and it also displayed suppression of GCN2 pathway activation with asparaginase treatment in the ALL cell line and mouse xenograft model.
RESUMO
Dysferlinopathies, which are muscular diseases caused by mutations in the dysferlin gene, remain serious medical problems due to the lack of therapeutic agents. Herein, we report the design, synthesis, and structure-activity relationships of a 2,6-disubstituted 3H-imidazo[4,5-b]pyridine series, which was identified from the phenotypic screening of chemicals that increase the level of dysferlin in myocytes differentiated from patient-derived induced pluripotent stem cells (iPSCs). Optimization studies with cell-based phenotypic assay led to the identification of a highly potent compound, 19, with dysferlin elevation effects at double-digit nanomolar concentrations. In addition, the molecular target of our chemical series was identified as tubulin, through a tubulin polymerization assay and a competitive binding assay using a photoaffinity labeling probe.
Assuntos
Imidazóis/química , Distrofia Muscular do Cíngulo dos Membros/tratamento farmacológico , Piridinas/química , Moduladores de Tubulina/uso terapêutico , Sítios de Ligação , Diferenciação Celular , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Disferlina/metabolismo , Células Hep G2 , Humanos , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Simulação de Acoplamento Molecular , Distrofia Muscular do Cíngulo dos Membros/patologia , Proteína MyoD/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Estrutura Terciária de Proteína , Piridinas/farmacologia , Piridinas/uso terapêutico , Relação Estrutura-Atividade , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologiaRESUMO
Chronic myelogenous leukemia (CML) is characterized by the oncogenic fusion protein, BCR-ABL protein kinase, against which clinically useful inhibitors have been developed. An alternative approach to treat CML is to degrade the BCR-ABL protein. Recently, potent degraders against BCR-ABL have been developed by conjugating dasatinib to ligands for E3 ubiquitin ligases. Since the degraders contain the dasatinib moiety, they also inhibit BCR-ABL kinase activity, which complicates our understanding of the impact of BCR-ABL degradation by degraders in CML growth inhibition. To address this issue, we chose DAS-IAP, as a potent BCR-ABL degrader, and developed a structurally related inactive degrader, DAS-meIAP, which inhibits kinase activity but does not degrade the BCR-ABL protein. DAS-IAP showed slightly weaker activity than DAS-meIAP in inhibiting cell growth when CML cells were treated for 48 h. However, DAS-IAP showed sustained growth inhibition even when the drug was removed after short-term treatment, whereas CML cell growth rapidly resumed following removal of DAS-meIAP and dasatinib. Consistently, suppression of BCR-ABL levels and downstream kinase signaling were maintained after DAS-IAP removal, whereas kinase signaling rapidly recovered following removal of DAS-meIAP and dasatinib. These results indicate that BCR-ABL degrader shows more sustained inhibition of CML cell growth than ABL kinase inhibitor.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Fusão bcr-abl/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Reagentes de Ligações Cruzadas , Dasatinibe/química , Dasatinibe/farmacologia , Dasatinibe/uso terapêutico , Desenho de Fármacos , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/genética , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Ligantes , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/uso terapêutico , Proteólise/efeitos dos fármacosRESUMO
Cyclin-dependent kinase 12 (CDK12) plays a key role in the coordination of transcription with elongation and mRNA processing. CDK12 mutations found in tumors and CDK12 inhibition sensitize cancer cells to DNA-damaging reagents and DNA-repair inhibitors. This suggests that CDK12 inhibitors are potential therapeutics for cancer that may cause synthetic lethality. Here, we report the discovery of 3-benzyl-1-( trans-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)-1-arylurea derivatives as novel and selective CDK12 inhibitors. Structure-activity relationship studies of a HTS hit, structure-based drug design, and conformation-oriented design using the Cambridge Structural Database afforded the optimized compound 2, which exhibited not only potent CDK12 (and CDK13) inhibitory activity and excellent selectivity but also good physicochemical properties. Furthermore, 2 inhibited the phosphorylation of Ser2 in the C-terminal domain of RNA polymerase II and induced growth inhibition in SK-BR-3 cells. Therefore, 2 represents an excellent chemical probe for functional studies of CDK12 and could be a promising lead compound for drug discovery.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Sobrevivência Celular , Quinases Ciclina-Dependentes/antagonistas & inibidores , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Inibidores Enzimáticos/química , Feminino , Humanos , Fosforilação , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Aberrant expression of proteins often underlies many diseases, including cancer. A recently developed approach in drug development is small molecule-mediated, selective degradation of dysregulated proteins. We have devised a protein-knockdown system that utilizes chimeric molecules termed specific and nongenetic IAP-dependent protein erasers (SNIPERs) to induce ubiquitylation and proteasomal degradation of various target proteins. SNIPER(ER)-87 consists of an inhibitor of apoptosis protein (IAP) ligand LCL161 derivative that is conjugated to the estrogen receptor α (ERα) ligand 4-hydroxytamoxifen by a PEG linker, and we have previously reported that this SNIPER efficiently degrades the ERα protein. Here, we report that derivatization of the IAP ligand module yields SNIPER(ER)s with superior protein-knockdown activity. These improved SNIPER(ER)s exhibited higher binding affinities to IAPs and induced more potent degradation of ERα than does SNIPER(ER)-87. Further, they induced simultaneous degradation of cellular inhibitor of apoptosis protein 1 (cIAP1) and delayed degradation of X-linked IAP (XIAP). Notably, these reengineered SNIPER(ER)s efficiently induced apoptosis in MCF-7 human breast cancer cells that require IAPs for continued cellular survival. We found that one of these molecules, SNIPER(ER)-110, inhibits the growth of MCF-7 tumor xenografts in mice more potently than the previously characterized SNIPER(ER)-87. Mechanistic analysis revealed that our novel SNIPER(ER)s preferentially recruit XIAP, rather than cIAP1, to degrade ERα. Our results suggest that derivatized IAP ligands could facilitate further development of SNIPERs with potent protein-knockdown and cytocidal activities against cancer cells requiring IAPs for survival.
Assuntos
Receptor alfa de Estrogênio/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Animais , Antineoplásicos/farmacologia , Regulação para Baixo , Humanos , Ligantes , Células MCF-7 , Camundongos , Ligação Proteica , Proteólise , Tiazóis/farmacologia , Ubiquitinação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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/químicaRESUMO
Protein degradation technology based on hybrid small molecules is an emerging drug modality that has significant potential in drug discovery and as a unique method of post-translational protein knockdown in the field of chemical biology. Here, we report the first example of a novel and potent protein degradation inducer that binds to an allosteric site of the oncogenic BCR-ABL protein. BCR-ABL allosteric ligands were incorporated into the SNIPER (Specific and Nongenetic inhibitor of apoptosis protein [IAP]-dependent Protein Erasers) platform, and a series of in vitro biological assays of binding affinity, target protein modulation, signal transduction, and growth inhibition were carried out. One of the designed compounds, 6 (SNIPER(ABL)-062), showed desirable binding affinities against ABL1, cIAP1/2, and XIAP and consequently caused potent BCR-ABL degradation.
RESUMO
The Ras proteins play roles in cell differentiation, proliferation, and survival. Aberrant signaling through Ras-mediated pathways in tumor cells occurs as a result of several types of mutational damage, which most frequently affects the amino acids G12, G13, and Q61. Recently, KRpep-2d was identified as a K-Ras(G12D) selective inhibitory peptide against the G12D mutant of K-Ras, which is a key member of the Ras protein family and an attractive cancer therapeutic target. In this study, the crystal structure of the human K-Ras(G12D) mutant was determined in complex with GDP and KRpep-2d at 1.25 Å resolution. This structure revealed that the peptide binds near Switch II and allosterically blocks protein-protein interactions with the guanine nucleotide exchange factor. This discovery of a unique binding pocket provides valuable information that will facilitate the design of direct Ras inhibitors.
RESUMO
Chromosomal translocation occurs in some cancer cells, which results in the expression of aberrant oncogenic fusion proteins that include BCR-ABL in chronic myelogenous leukemia (CML). Inhibitors of ABL tyrosine kinase, such as imatinib and dasatinib, exhibit remarkable therapeutic effects, although emergence of drug resistance hampers the therapy during long-term treatment. An alternative approach to treat CML is to downregulate the BCR-ABL protein. We have devised a protein knockdown system by hybrid molecules named Specific and Non-genetic inhibitor of apoptosis protein [IAP]-dependent Protein Erasers (SNIPER), which is designed to induce IAP-mediated ubiquitylation and proteasomal degradation of target proteins, and a couple of SNIPER(ABL) against BCR-ABL protein have been developed recently. In this study, we tested various combinations of ABL inhibitors and IAP ligands, and the linker was optimized for protein knockdown activity of SNIPER(ABL). The resulting SNIPER(ABL)-39, in which dasatinib is conjugated to an IAP ligand LCL161 derivative by polyethylene glycol (PEG) × 3 linker, shows a potent activity to degrade the BCR-ABL protein. Mechanistic analysis suggested that both cellular inhibitor of apoptosis protein 1 (cIAP1) and X-linked inhibitor of apoptosis protein (XIAP) play a role in the degradation of BCR-ABL protein. Consistent with the degradation of BCR-ABL protein, the SNIPER(ABL)-39 inhibited the phosphorylation of signal transducer and activator of transcription 5 (STAT5) and Crk like proto-oncogene (CrkL), and suppressed the growth of BCR-ABL-positive CML cells. These results suggest that SNIPER(ABL)-39 could be a candidate for a degradation-based novel anti-cancer drug against BCR-ABL-positive CML.
Assuntos
Dasatinibe/farmacologia , Proteínas de Fusão bcr-abl/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Tiazóis/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dasatinibe/química , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células K562 , Ligantes , Fosforilação/efeitos dos fármacos , Ligação Proteica , Inibidores de Proteínas Quinases/química , Proteólise , Proto-Oncogene Mas , Ubiquitinação/efeitos dos fármacosRESUMO
A structure-activity relationship study of a K-Ras(G12D) selective inhibitory cyclic peptide, KRpep-2d was performed. Alanine scanning of KRpep-2d focusing on the cyclic moiety showed that Leu7, Ile9, and Asp12 are the key elements for K-Ras(G12D) selective inhibition of KRpep-2d. The cysteine bridging was also examined to identify the stable analog of KRpep-2d under reductive conditions. As a result, the KRpep-2d analog (12) including mono-methylene bridging showed potent K-Ras(G12D) selective inhibition in both the presence and the absence of dithiothreitol. This means that mono-methylene bridging is an effective strategy to obtain a reduction-resistance analog of parent disulfide cyclic peptides. Peptide 12 inhibited proliferation of K-Ras(G12D)-driven cancer cells significantly. These results gave valuable information for further optimization of KRpep-2d to provide novel anti-cancer drug candidates targeting the K-Ras(G12D) mutant.
Assuntos
Alanina/farmacologia , Antineoplásicos/farmacologia , Cisteína/farmacologia , Peptídeos Cíclicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Alanina/química , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisteína/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Mutação , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/química , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Relação Estrutura-AtividadeRESUMO
Recently, there have been a limited number of new, validated targets for small-molecule drug discovery in the pharmaceutical industry. Although there are approximately 30â¯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/químicaRESUMO
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.
RESUMO
Amino-acid mutations of Gly12 (e.g. G12D, G12V, G12C) of V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras), the most promising drug target in cancer therapy, are major growth drivers in various cancers. Although over 30 years have passed since the discovery of these mutations in most cancer patients, effective mutated K-Ras inhibitors have not been marketed. Here, we report novel and selective inhibitory peptides to K-Ras(G12D). We screened random peptide libraries displayed on T7 phage against purified recombinant K-Ras(G12D), with thorough subtraction of phages bound to wild-type K-Ras, and obtained KRpep-2 (Ac-RRCPLYISYDPVCRR-NH2) as a consensus sequence. KRpep-2 showed more than 10-fold binding- and inhibition-selectivity to K-Ras(G12D), both in SPR analysis and GDP/GTP exchange enzyme assay. KD and IC50 values were 51 and 8.9 nM, respectively. After subsequent sequence optimization, we successfully generated KRpep-2d (Ac-RRRRCPLYISYDPVCRRRR-NH2) that inhibited enzyme activity of K-Ras(G12D) with IC50 = 1.6 nM and significantly suppressed ERK-phosphorylation, downstream of K-Ras(G12D), along with A427 cancer cell proliferation at 30 µM peptide concentration. To our knowledge, this is the first report of a K-Ras(G12D)-selective inhibitor, contributing to the development and study of K-Ras(G12D)-targeting drugs.
Assuntos
Descoberta de Drogas/métodos , Neoplasias Experimentais/tratamento farmacológico , Biblioteca de Peptídeos , Inibidores de Proteases/administração & dosagem , Inibidores de Proteases/química , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Bacteriófago T7 , Sítios de Ligação , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Humanos , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Inibidores de Proteases/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas/métodos , Proteínas Proto-Oncogênicas p21(ras)/metabolismoRESUMO
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 TempoRESUMO
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 & inibidoresRESUMO
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/farmacologiaRESUMO
Controversy exists over whether the chaperonin GroEL forms a GroEL-(GroES)2 complex (football-shaped complex) during its reaction cycle. We have revealed previously the existence of the football-shaped complex in the chaperonin reaction cycle using a FRET (fluorescence resonance energy transfer) assay [Sameshima, Ueno, Iizuka, Ishii, Terada, Okabe and Funatsu (2008) J. Biol. Chem. 283, 23765-23773]. Although denatured proteins alter the ATPase activity of GroEL and the dynamics of the GroEL-GroES interaction, the effect of denatured proteins on the formation of the football-shaped complex has not been characterized. In the present study, a FRET assay was used to demonstrate that denatured proteins facilitate the formation of the football-shaped complex. The presence of denatured proteins was also found to increase the rate of association of GroES to the trans-ring of GroEL. Furthermore, denatured proteins decrease the inhibitory influence of ADP on ATP-induced association of GroES to the trans-ring of GroEL. From these findings we conclude that denatured proteins facilitate the dissociation of ADP from the trans-ring of GroEL and the concomitant association of ATP and the second GroES.
Assuntos
Chaperonina 10/química , Chaperonina 60/química , Complexos Multiproteicos/química , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Chaperonina 10/metabolismo , Chaperonina 60/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , Cinética , Ligação Proteica , Conformação Proteica , Desnaturação ProteicaRESUMO
GroEL is an Escherichia coli chaperonin that is composed of two heptameric rings stacked back-to-back. GroEL assists protein folding with its cochaperonin GroES in an ATP-dependent manner in vitro and in vivo. However, it is still unclear whether GroES binds to both rings of GroEL simultaneously under physiological conditions. In this study, we monitored the GroEL-GroES interaction in the reaction cycle using fluorescence resonance energy transfer. We found that nearly equivalent amounts of symmetric GroEL-(GroES)(2) (football-shaped) complex and asymmetric GroEL-GroES (bullet-shaped) complex coexist during the functional reaction cycle. We also found that D398A, an ATP hydrolysis defective mutant of GroEL, forms a football-shaped complex with ATP bound to the two rings. Furthermore, we showed that ADP prevents the association of ATP to the trans-ring of GroEL, and as a consequence, the second GroES cannot bind to GroEL. Considering the concentrations of ADP and ATP in E. coli, ADP is expected to have a small effect on the inhibition of GroES binding to the trans-ring of GroEL in vivo. These results suggest that we should reconsider the chaperonin-mediated protein-folding mechanism that involves the football-shaped complex.