RESUMO
Extracellular HSP90α (eHSP90α) is a promoter of tumor development and malignant progression. Patients with malignancies, including pancreatic ductal adenocarcinoma (PDAC), have generally shown 5~10-fold increases in serum/plasma eHSP90α levels. In this study, we developed a humanized antibody HH01 to target eHSP90α and evaluated its anticancer efficacy. HH01, with novel complementarity-determining regions, exhibits high binding affinity toward HSP90α. It recognizes HSP90α epitope sites 235AEEKEDKEEE244 and 251ESEDKPEIED260, with critical amino acid residues E237, E239, D240, K241, E253, and K255. HH01 effectively suppressed eHSP90α-induced invasive and spheroid-forming activities of colorectal cancer and PDAC cell lines by blocking eHSP90α's ligation with the cell-surface receptor CD91. In mouse models, HH01 potently inhibited the tumor growth of PDAC cell grafts/xenografts promoted by endothelial-mesenchymal transition-derived cancer-associated fibroblasts while also reducing serum eHSP90α levels, reflecting its anticancer efficacy. HH01 also modulated tumor immunity by reducing M2 macrophages and reinvigorating immune T-cells. Additionally, HH01 showed low aggregation propensity, high water solubility, and a half-life time of >18 days in mouse blood. It was not cytotoxic to retinal pigmented epithelial cells and showed no obvious toxicity in mouse organs. Our data suggest that targeting eHSP90α with HH01 antibody can be a promising novel strategy for PDAC therapy.
Assuntos
Anticorpos Monoclonais Humanizados , Proteínas de Choque Térmico HSP90 , Neoplasias Pancreáticas , Humanos , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Animais , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , Camundongos , Linhagem Celular Tumoral , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Adenocarcinoma/patologia , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/metabolismo , Transição Endotélio-MesênquimaRESUMO
The coronavirus (CoV) disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a health threat worldwide. Viral main protease (Mpro, also called 3C-like protease [3CLpro]) is a therapeutic target for drug discovery. Herein, we report that GC376, a broad-spectrum inhibitor targeting Mpro in the picornavirus-like supercluster, is a potent inhibitor for the Mpro encoded by SARS-CoV-2, with a half-maximum inhibitory concentration (IC50) of 26.4 ± 1.1 nM. In this study, we also show that GC376 inhibits SARS-CoV-2 replication with a half-maximum effective concentration (EC50) of 0.91 ± 0.03 µM. Only a small portion of SARS-CoV-2 Mpro was covalently modified in the excess of GC376 as evaluated by mass spectrometry analysis, indicating that improved inhibitors are needed. Subsequently, molecular docking analysis revealed that the recognition and binding groups of GC376 within the active site of SARS-CoV-2 Mpro provide important new information for the optimization of GC376. Given that sufficient safety and efficacy data are available for GC376 as an investigational veterinary drug, expedited development of GC376, or its optimized analogues, for treatment of SARS-CoV-2 infection in human is recommended.
Assuntos
Antivirais/química , Betacoronavirus/efeitos dos fármacos , Cisteína Endopeptidases/química , Inibidores de Proteases/química , Pirrolidinas/química , Proteínas não Estruturais Virais/química , Motivos de Aminoácidos , Animais , Antivirais/farmacologia , Betacoronavirus/patogenicidade , Domínio Catalítico , Chlorocebus aethiops , Proteases 3C de Coronavírus , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Expressão Gênica , Simulação de Acoplamento Molecular , Inibidores de Proteases/farmacologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Pirrolidinas/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SARS-CoV-2 , Ácidos Sulfônicos , Termodinâmica , Células Vero , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Zika virus (ZIKV) of the flaviviridae family, is the cause of emerging infections characterized by fever, Guillain-Barré syndrome (GBS) in adults and microcephaly in newborns. There exists an urgent unmet clinical need for anti-ZIKV drugs for the treatment of infected individuals. In the current work, we aimed at the promising virus drug target, ZIKV NS3 protease and constructed a Pharmacophore Anchor (PA) model for the active site. The PA model reveals a total of 12 anchors (E, H, V) mapped across the active site subpockets. We further identified five of these anchors to be critical core anchors (CEH1, CH3, CH7, CV1, CV3) conserved across flaviviral proteases. The ZIKV protease PA model was then applied in anchor-enhanced virtual screening yielding 14 potential antiviral candidates, which were tested by in vitro assays. We discovered FDA drugs Asunaprevir and Simeprevir to have potent anti-ZIKV activities with EC50 values 4.7 µM and 0.4 µM, inhibiting the viral protease with IC50 values 6.0 µM and 2.6 µM respectively. Additionally, the PA model anchors aided in the exploration of inhibitor binding mechanisms. In conclusion, our PA model serves as a promising guide map for ZIKV protease targeted drug discovery and the identified 'previr' FDA drugs are promising for anti-ZIKV treatments.
Assuntos
Antivirais/farmacologia , Descoberta de Drogas/métodos , Serina Endopeptidases/efeitos dos fármacos , Proteínas Virais/efeitos dos fármacos , Zika virus/efeitos dos fármacos , Domínio Catalítico/efeitos dos fármacos , Modelos Químicos , Simulação de Acoplamento Molecular , Alinhamento de Sequência , Zika virus/enzimologia , Zika virus/genéticaRESUMO
In the past few decades, enterovirus A71 (EVA71) has caused devastating outbreaks in the Asia-Pacific region, resulting in serious sequelae in infected young children. No preventive or therapeutic interventions are currently available for curing EVA71 infection, highlighting a great unmet medical need for this disease. Here, we showed that one novel single-domain antibody (sdAb), F1, isolated from an immunized llama, could alleviate EVA71 infection both in vitro and in vivo We also confirmed that the sdAb clone F1 recognizes EVA71 through a novel conformational epitope comprising the highly conserved region of VP3 capsid protein by using competitive-binding and overlapping-peptide enzyme-linked immunosorbent assays (ELISAs). Because of the virion's icosahedral structure, we reasoned that adjacent epitopes must be clustered within molecular ranges that may be simultaneously bound by an engineered antibody with multiple valency. Therefore, two single-domain binding modules (F1) were fused to generate an sdAb-in-tandem design so that the capture of viral antigens could be further increased by valency effects. We showed that the tetravalent construct F1×F1-hFc, containing two sdAb-in-tandem on a fragment crystallizable (Fc) scaffold, exhibits more potent neutralization activity against EVA71 than does the bivalent sdAb F1-hFc by at least 5.8-fold. We also demonstrated that, using a human scavenger receptor class B member 2 (hSCARB2) transgenic mouse model, a half dose of the F1×F1-hFc provided better protection against EVA71 infection than did the F1-hFc. Thus, our study furnishes important insights into multivalent sdAb engineering against viral infection and provides a novel strategic deployment approach for preparedness of emerging infectious diseases such as EVA71.
Assuntos
Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/imunologia , Enterovirus Humano A/imunologia , Infecções por Enterovirus/terapia , Anticorpos de Domínio Único/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/farmacologia , Antígenos Virais/imunologia , Camelídeos Americanos , Linhagem Celular Tumoral , Infecções por Enterovirus/imunologia , Infecções por Enterovirus/prevenção & controle , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Camundongos , Camundongos Transgênicos , Anticorpos de Domínio Único/farmacologiaRESUMO
Atomic adsorption of Au and Pt on the rutile (1 1 0) surface was investigated by atomic-resolution aberration-corrected scanning transmission electron microscopy (STEM) measurements combined with density functional theory calculations. Au single atoms were deposited on the surface in a vacuum condition, and the observed results were compared with Pt single atoms on the same surface prepared by the same experimental manner. It was found that Au single atoms are stably adsorbed only at the bridging oxygen vacancy sites, which is quite different from Pt single atoms exhibiting the most frequently observed adsorption at the basal oxygen vacancy sites. Such a difference in oxygen-vacancy effect between Au and Pt can be explained by electronic structures of the surface vacancies as well as characters of outermost atomic orbitals of Au and Pt.
RESUMO
The overexpression of HER2/neu and EGFR receptors plays important roles in tumorigenesis and tumor progression. Targeting these two receptors simultaneously can have a more widespread application in early diagnosis of cancers. In this study, a new multifunctional nanoparticles (MnMEIO-CyTE777-(Bis)-mPEG NPs) comprising a manganese-doped iron oxide nanoparticle core (MnMEIO), a silane-amino functionalized poly(ethylene glycol) copolymer shell, a near infrared fluorescence dye (CyTE777), and a covalently conjugated anti-HER2/neu and anti-EGFR receptors bispecific antibody (Bis) were successfully developed. In vitro T2-weighted MR imaging studies in SKBR-3 and A431 tumor cells incubated with MnMEIO-CyTE777-(Bis)-mPEG NPs showed - 94.8 ± 3.8 and - 84.1 ± 2.8% negative contrast enhancement, respectively. Pharmacokinetics study showed that MnMEIO-CyTE777-(Bis)-mPEG NPs were eliminated from serum with the half-life of 21.3 mins. In vivo MR imaging showed that MnMEIO-CyTE777-(Bis)-mPEG NPs could specifically and effectively target to HER2/neu- and EGFR-expressing tumors in mice; the relative contrast enhancements were 11.8 (at 2 hrs post-injection) and 61.5 (at 24 hrs post-injection) fold higher in SKBR-3 tumors as compared to Colo-205 tumors. T2-weighted MR and optical imaging studies revealed that the new contrast agent (MnMEIO-CyTE777-(Bis)-mPEG NPs) could specifically and effectively target to HER2/neu- and/or EGFR-expressing tumors. Our results demonstrate that MnMEIO-CyTE777-(Bis)-mPEG NPs are able to recognize the tumors expressing both HER2/neu and/or EGFR, and may provide a novel molecular imaging tool for early diagnosis of cancers expressing HER2/neu and/or EGFR.
Assuntos
Anticorpos Biespecíficos/farmacocinética , Receptores ErbB/análise , Compostos Férricos/farmacocinética , Imageamento por Ressonância Magnética/métodos , Manganês/farmacocinética , Neoplasias/diagnóstico , Receptor ErbB-2/análise , Animais , Magnetismo , Camundongos , Neoplasias/patologia , Coloração e Rotulagem/métodosRESUMO
Enterovirus 71 (EV-A71) is a neurotropic virus that can cause severe complications involving the central nervous system. No effective antiviral therapeutics are available for treating EV-A71 infection and drug discovery efforts are rarely focused to target this disease. Thus, the main goal of this study was to discover existing drugs with novel indications that may effectively inhibit EV-A71 replication and the inflammatory cytokines elevation. In this study, we showed that LiCl, a GSK3ß inhibitor, effectively suppressed EV-A71 replication, apoptosis and inflammatory cytokines production (Interleukin 6, Interleukin-1ß) in infected cells. Furthermore, LiCl and an immunomodular agent were shown to strongly synergize with each other in suppressing EV-A71 replication. The results highlighted potential new treatment regimens in suppressing sequelae caused by EV-A71 replication.
Assuntos
Compostos de Anilina/administração & dosagem , Antivirais/administração & dosagem , Enterovirus Humano A/efeitos dos fármacos , Infecções por Enterovirus/tratamento farmacológico , Hidroxibutiratos/administração & dosagem , Cloreto de Lítio/administração & dosagem , Compostos de Anilina/farmacologia , Animais , Antivirais/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Chlorocebus aethiops , Crotonatos , Citocinas/genética , Citocinas/metabolismo , Reposicionamento de Medicamentos , Sinergismo Farmacológico , Quimioterapia Combinada , Enterovirus Humano A/fisiologia , Infecções por Enterovirus/imunologia , Humanos , Hidroxibutiratos/farmacologia , Cloreto de Lítio/farmacologia , Nitrilas , Toluidinas , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
Noble metal nanoparticles (e.g., gold and platinum) supported on TiO2 surfaces are utilized in many technological applications such as heterogeneous catalysts. To fully understand their enhanced catalytic activity, it is essential to unravel the interfacial interaction between the metal atoms and TiO2 surfaces at the level of atomic dimensions. However, it has been extremely difficult to directly characterize the atomic-scale structures that result when individual metal atoms are adsorbed on the TiO2 surfaces. Here, we show direct atomic-resolution images of individual Pt atoms adsorbed on TiO2 (110) surfaces using aberration-corrected scanning transmission electron microscopy. Subangstrom spatial resolution enables us to identify five different Pt atom adsorption sites on the TiO2 (110) surface. Combining this with systematic density functional theory calculations reveals that the most favorable Pt adsorption sites are on vacancy sites of basal oxygen atoms that are located in subsurface positions relative to the top surface bridging oxygen atoms.
RESUMO
BACKGROUND: There are few studies that have examined the potential of RNA inference (RNAi) to increase protein production in the baculovirus expression vector system (BEVS). Spodoptera frugiperda (fall armyworm) (Sf)-caspase-1-repressed stable cells exhibit resistance to apoptosis and enhancement of recombinant protein production. However, the mechanism of recombinant protein augmentation in baculovirus-infected Caspase-repressed insect cells has not been elucidated. RESULTS: In the current study, we utilized RNAi-mediated Sf-caspase-1-repressed stable cells to clarify how the resistance to apoptosis can enhance both intracellular (firefly luciferase) and extracellular (secreted alkaline phosphatase [SEAP]) recombinant protein production in BEVS. Since the expression of molecular chaperones is strongly associated with the maximal production of exogenous proteins in BEVS, the differential expression of molecular chaperones in baculovirus-infected stable cells was also analyzed in this study. CONCLUSION: The data indicated that the retention of expression of molecular chaperones in baculovirus-infected Sf-caspase-1-repressed stable cells give the higher recombinant protein accumulation.
Assuntos
Caspases/metabolismo , Chaperonas Moleculares/metabolismo , Animais , Baculoviridae/genética , Caspases/química , Caspases/genética , Chaperonas Moleculares/genética , Plasmídeos/genética , Plasmídeos/metabolismo , Interferência de RNA , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Células Sf9 , Spodoptera , TransfecçãoRESUMO
An acquired mutation (T790M) in the epidermal growth factor receptor (EGFR) accounts for half of all relapses in non-small cell lung cancer (NSCLC) patients who initially respond to EGFR kinase inhibitors. In this study, we demonstrated for the first time that EGFR-T790M interacts with the cytoskeletal components, myosin heavy chain 9 (MYH9) and ß-actin, in the nucleus of H1975 cells carrying the T790M-mutant EGFR. The interactions of EGFR with MYH9 and ß-actin were reduced in the presence of blebbistatin, a specific inhibitor for the MYH9-ß-actin interaction, suggesting that the EGFR interaction with MYH9 and ß-actin is affected by the integrity of the cytoskeleton. These physical interactions among MYH9, ß-actin, and EGFR were also impaired by CL-387,785, a kinase inhibitor for EGFR-T790M. Furthermore, CL-387,785 and blebbistatin interacted in a synergistic fashion to suppress cell proliferation and induce apoptosis in H1975 cells. The combination of CL-387,785 and blebbistatin enhanced the down-regulation of cyclooxygenase-2 (COX-2), a transcriptional target of nuclear EGFR. Overall, our findings demonstrate that disrupting EGFR interactions with the cytoskeletal components enhanced the anti-cancer effects of CL-387,785 against H1975 cells, suggesting a novel therapeutic approach for NSCLC cells that express the drug-resistant EGFR-T790M.
Assuntos
Receptores ErbB/metabolismo , Proteínas Motores Moleculares/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo II/metabolismo , Actinas/metabolismo , Apoptose/genética , Apoptose/fisiologia , Western Blotting , Linhagem Celular , Núcleo Celular/metabolismo , Proliferação de Células , Ciclo-Oxigenase 2/metabolismo , Citometria de Fluxo , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Imunoprecipitação , Miosina Tipo II/antagonistas & inibidores , Ligação Proteica , Quinazolinas/farmacologia , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo RealRESUMO
A high-throughput 32D(L858R/T790M) cell-based assay to identify inhibitors of the L858R/T790M mutant epidermal growth factor receptor (EGFR) pathway was established. After screening, ten hits from among 60,000 compounds in our in-house compound library were initially identified. In the secondary assays, one hit, 1-[2-(decyloxy)-2-oxoethyl]-3-methyl-2-[(4-methylphenoxy) methyl]-1H-benzimidazol-3-ium, was confirmed to directly inhibit the kinase activity of recombinant L858R/T790M EGFR and the phosphorylation of EGFR-L858R/T790M in gefitinib-resistant H1975 cells. Thus, this high-throughput assay system may be useful for identifying novel inhibitors which suppress mutant EGFR-T790M signalling and for overcoming T790M-mediated acquired resistance for future anticancer drug discovery.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Neoplasias Pulmonares/tratamento farmacológico , Mutação/efeitos dos fármacos , Quinazolinas/farmacologia , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Gefitinibe , Ensaios de Triagem em Larga Escala , Humanos , Neoplasias Pulmonares/genética , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Diosgenin (3ß-hydroxy-5-spirostene, 1), a plant-derived sapogenin, is used as a dietary supplement. However, the biological effects of 1 related to viral replication remain unexplored. In this study, the effects of 1 on hepatitis C virus (HCV) replication were evaluated. Based on a reporter-based HCV subgenomic replicon system, 1 was found to inhibit HCV replication at low micromolar concentrations. The EC(50) (concentration at which 50% of HCV replication is inhibited) of 1 was 3.8 µM. No cellular toxicity was observed at this concentration. Diosgenin (1) also significantly reduced the levels of viral RNA and viral proteins as evaluated by quantitative real-time reverse transcriptase PCR and Western blot analysis, respectively. In addition, in an alternative HCV antiviral system more closely aligned to all steps involved in the HCV infection and life cycle, 1 totally abolished HCV replication at 20 µM. Moreover, 1 reduced the phosphorylation of signal transducer and activator of transcription 3. A combination of 1 and interferon-α exerted an additive effect on the resultant anti-HCV activity.
Assuntos
Antivirais/farmacologia , Diosgenina/farmacologia , Hepacivirus/efeitos dos fármacos , Sapogeninas/farmacologia , Antivirais/química , Antivirais/isolamento & purificação , Suplementos Nutricionais/análise , Diosgenina/química , Diosgenina/isolamento & purificação , Hepacivirus/genética , Humanos , Estrutura Molecular , RNA Viral/análise , RNA Viral/efeitos dos fármacos , Sapogeninas/química , Sapogeninas/isolamento & purificação , Proteínas não Estruturais Virais/efeitos dos fármacosRESUMO
HTS hit 7 was modified through hybrid design strategy to introduce a chiral side chain followed by introduction of Michael acceptor group to obtain potent EGFR kinase inhibitors 11 and 19. Both 11 and 19 showed over 3 orders of magnitude enhanced HCC827 antiproliferative activity compared to HTS hit 7 and also inhibited gefitinib-resistant double mutant (DM, T790M/L858R) EGFR kinase at nanomolar concentration. Moreover, treatment with 19 shrinked tumor in nude mice xenograft model.
Assuntos
Antineoplásicos/síntese química , Receptores ErbB/antagonistas & inibidores , Compostos Heterocíclicos com 3 Anéis/síntese química , Pirimidinas/síntese química , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas , Linhagem Celular Tumoral , Desenho de Fármacos , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Receptores ErbB/genética , Gefitinibe , Compostos Heterocíclicos com 3 Anéis/química , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Masculino , Camundongos , Camundongos Nus , Mutação , Transplante de Neoplasias , Pirimidinas/química , Pirimidinas/farmacologia , Quinazolinas/farmacologia , Ratos , Ratos Sprague-Dawley , Estereoisomerismo , Relação Estrutura-Atividade , Transplante HeterólogoRESUMO
Herein we reveal a simple method for the identification of novel Aurora kinase A inhibitors through substructure searching of an in-house compound library to select compounds for testing. A hydrazone fragment conferring Aurora kinase activity and heterocyclic rings most frequently reported in kinase inhibitors were used as substructure queries to filter the in-house compound library collection prior to testing. Five new series of Aurora kinase inhibitors were identified through this strategy, with IC(50) values ranging from approximately 300 nM to approximately 15 microM, by testing only 133 compounds from a database of approximately 125,000 compounds. Structure-activity relationship studies and X-ray co-crystallographic analysis of the most potent compound, a furanopyrimidine derivative with an IC(50) value of 309 nM toward Aurora kinase A, were carried out. The knowledge gained through these studies could help in the future design of potent Aurora kinase inhibitors.
Assuntos
Furanos/química , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirimidinas/química , Aurora Quinases , Sítios de Ligação , Cristalografia por Raios X , Ensaios de Triagem em Larga Escala , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Pirimidinas/síntese química , Pirimidinas/farmacologia , Relação Estrutura-AtividadeRESUMO
3-Mercapto-1-propanesulfonic acid (MPS) and bis(3-sulfopropyl) disulfide (SPS) adsorbed on a Au(111) electrode were studied by using in situ scanning tunneling microscopy (STM). Although the adsorptions of MPS and SPS are known to be oxidative and reductive, respectively, on an Au(111) electrode, these two admolecules behave similarly in terms of phase evolution, surface coverage, potential for stripping, and characteristics of cyclic voltammetry. However, different adsorption mechanisms of these molecules result in different structures. Raising electrode potential causes more MPS and SPS molecules to adsorb, yielding ordered adlattices between 0.67 and 0.8 V (vs reversible hydrogen electrode). The ordered adlattices of MPS and SPS appear as striped and netlike structures with molecules adsorbed parallel to the Au(111) surface. Switching potential to 0.9 V or more positive still does not result in upright molecular orientation, possibly inhibited by electrostatic interaction between the end group of -SO(3)(-) and the Au(111) electrode. Lowering the potential to 0.4 V disrupted the ordered adlayer. Stripping voltammetric experiments show that MPS and SPS admolecules are desorbed from Au(111) at the same potential, suggesting that these molecules are both adsorbed via their sulfur headgroups. The S-S bond in SPS is likely broken upon its adsorption on Au(111).
RESUMO
Epidermal growth factor receptor (EGFR) is a valid drug target for development of target-based therapeutics against non-small-cell lung cancer. In this study, we established a high-throughput cell-based assay to screen for compounds that may inhibit EGFR activation and/or EGFR-mediated downstream signaling pathway. This drug screening platform is based on the characterization of an EGFR-transfected 32D cell line (32D-EGFR). The expression of EGFR in 32D cells allowed cell proliferation in the presence of either epidermal growth factor (EGF) or interleukin 3 (IL-3) and provided a system for both screening and counterscreening of EGFR pathway-inhibitory compounds. After the completion of primary and secondary screenings in which 32D-EGFR cells were grown under the stimulation of either EGF or IL-3, 9 of 20,000 compounds were found to selectively inhibit the EGF-dependent proliferation, but not the IL-3-dependent proliferation, of 32D-EGFR cells. Subsequent analysis showed that 3 compounds of the 9 initial hits directly inhibited the kinase activity of recombinant EGFR in vitro and the phosphorylation of EGFR in H1299 cells transfected with EGFR. Thus, this 32D-EGFR assay system provides a promising approach for identifying novel EGFR and EGFR signaling pathway inhibitors with potential antitumor activity.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Humanos , Camundongos , Fosforilação/efeitos dos fármacos , Reprodutibilidade dos TestesRESUMO
Tricyclic 6,7-dihydro-4H-pyrazolo[1,5-a]pyrrolo[3,4-d]pyrimidine-5,8-dione was identified as a novel scaffold for Aurora kinase A inhibition through virtual screening. SAR exploration coupled with molecular modeling of 8a reveals the minimum pharmacophore requirements for Aurora kinase A inhibition.
Assuntos
Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirazóis/química , Pirimidinonas/química , Pirróis/química , Aurora Quinases , Sítios de Ligação , Modelos Moleculares , Estrutura Molecular , Proteínas Serina-Treonina Quinases/química , Relação Estrutura-AtividadeRESUMO
A potent SARS coronavirus (CoV) 3CL protease inhibitor (TG-0205221, Ki = 53 nM) has been developed. TG-0205221 showed remarkable activity against SARS CoV and human coronavirus (HCoV) 229E replications by reducing the viral titer by 4.7 log (at 5 microM) for SARS CoV and 5.2 log (at 1.25 microM) for HCoV 229E. The crystal structure of TG-0205221 (resolution = 1.93 A) has revealed a unique binding mode comprising a covalent bond, hydrogen bonds, and numerous hydrophobic interactions. Structural comparisons between TG-0205221 and a natural peptide substrate were also discussed. This information may be applied toward the design of other 3CL protease inhibitors.