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To understand why Chlamydia trachomatis (Ct) (L2/434/Bu) favors hypoxia, we examined the dynamics of infected cells using a glycolysis-related PCR array and metabolomic analysis, along with the perturbation of nucleotide synthesis. Our findings revealed that, compared to normoxia, hypoxia with infection significantly and selectively upregulates the expression of genes related to glycolysis, glycogen degradation, and the pentose phosphate pathway. Furthermore, hypoxia induced a significant decrease in metabolite levels, particularly methionine-related metabolites, independent of infection, indicating efficient metabolism under hypoxia. Additionally, the perturbation of nucleotide synthesis with adenosine derivatives impaired Ct growth. Collectively, our results suggest that Ct favors a hypoxic environment with efficient metabolism, in which Ct readily activates glycolysis responsible for stable nucleotide synthesis as well as ATP supply.
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Small antibody fragments have recently been used as alternatives to full-length monoclonal antibodies in therapeutic applications. One of the most popular fragment antibodies is single-chain fragment variables (scFvs), consisting of variable heavy (VH) and variable light (VL) domains linked by a flexible peptide linker. scFvs have small molecular sizes, which enables good tissue penetration and low immunogenicity. Despite these advantages, the use of scFvs, especially for therapeutic purpose, is still limited because of the difficulty to regulate the binding activity and conformational stability. In this study, we constructed and analyzed 10 scFv fragments derived from 10 representatives of FDA-approved mAbs to evaluate their physicochemical properties. Differential scanning calorimetry analysis showed that scFvs exhibited relatively high but varied thermostability, from 50 to 70°C of melting temperatures, and different unfolding cooperativity. Surface plasmon resonance analysis revealed that scFvs fragments that exhibit high stability and cooperative unfolding likely tend to maintain antigen binding. This study demonstrated the comprehensive physicochemical properties of scFvs derived from FDA-approved antibodies, providing insights into antibody design and development.
Assuntos
Estabilidade Proteica , Anticorpos de Cadeia Única , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/imunologia , Humanos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Varredura Diferencial de Calorimetria , Ligação ProteicaRESUMO
Researchers collect data and use various methods to organize it. Ensuring the reliability and reproducibility of data is crucial, and collaboration across different research fields is on the rise. However, when there is geographical distance, sharing data becomes a challenging task. Therefore, there is a need for the development of a mechanism for sharing data on the web. We have developed an integrated database to facilitate the sharing and management of research data, particularly focusing on small molecules. The integrated database serves as a platform for centralizing data related to small molecules, including their chemical structures, wet lab experimental data, simulation data, and more. It has been constructed as a web application, offering features such as library management for small molecules, registration and viewing of wet lab experiment results, generation of initial conformations for simulations, and data visualization. This enables researchers to efficiently share their research data and collaborate seamlessly, whether within their research group or via cloud-based access that allows project and team members to connect from anywhere. This integrated database plays a critical role in connecting wet lab experiments and simulations, enabling researchers to cross-reference and analyze experimental data comprehensively. It serves as an essential tool to advance research and foster idea generation.
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Bases de Dados Factuais , Simulação por Computador , Disseminação de Informação , Internet , Reprodutibilidade dos Testes , Bibliotecas de Moléculas PequenasRESUMO
Dihydromaniwamycin E (1), a new maniwamycin derivative featuring an azoxy moiety, has been isolated from the culture extract of thermotolerant Streptomyces sp. JA74 along with the known analogue maniwamycin E (2). Compound 1 is produced only by cultivation of strain JA74 at 45 °C, and this type of compound has been previously designated a "heat shock metabolite (HSM)" by our research group. Compound 2 is detected as a production-enhanced metabolite at high temperature. Structures of 1 and 2 are elucidated by NMR and MS spectroscopic analyses. The absolute structure of 1 is determined after the total synthesis of four stereoisomers. Though the absolute structure of 2 has been proposed to be the same as the structure of maniwamycin D, the NMR and the optical rotation value of 2 are in agreement with those of maniwamycin E. Therefore, this study proposes a structural revision of maniwamycins D and E. Compounds 1 and 2 show inhibitory activity against the influenza (H1N1) virus infection of MDCK cells, demonstrating IC50 values of 25.7 and 63.2 µM, respectively. Notably, 1 and 2 display antiviral activity against SARS-CoV-2, the causative agent of COVID-19, when used to infect 293TA and VeroE6T cells, with 1 and 2 showing IC50 values (for infection of 293TA cells) of 19.7 and 9.7 µM, respectively. The two compounds do not exhibit cytotoxicity in these cell lines at those IC50 concentrations.
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Antivirais , Compostos Azo , COVID-19 , Vírus da Influenza A Subtipo H1N1 , SARS-CoV-2 , Streptomyces , Humanos , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Compostos Azo/química , Compostos Azo/metabolismo , Compostos Azo/farmacologia , Resposta ao Choque Térmico , Células HEK293 , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Influenza Humana/tratamento farmacológico , Células Madin Darby de Rim Canino , Infecções por Orthomyxoviridae/tratamento farmacológico , SARS-CoV-2/efeitos dos fármacos , Streptomyces/química , Streptomyces/metabolismo , Células Vero , Chlorocebus aethiops , CãesRESUMO
Because the breakdown of the blood-brain spinal cord barrier (BBSCB) worsens many central nervous system (CNS) diseases, prevention of BBSCB breakdown has been a major therapeutic target, especially for spinal cord injury (SCI). However, effective drugs that protect BBSCB function have yet to be developed. The purpose of the current study was 1) to develop a high-throughput screening assay (HTSA) to identify candidate drugs to protect BBSCB function, 2) to identify candidate drugs from existing drugs with newly developed HTSA, and 3) to examine the therapeutic effects of candidate drugs on SCI. Our HTSA included a culture of immortalized human brain endothelial cells primed with candidate drugs, stress with H2O2, and evaluation of their viability. A combination of the resazurin-based assay with 0.45 mM H2O2 qualified as a reliable HTSA. Screening of 1,570 existing drugs identified 90 drugs as hit drugs. Through a combination of reproducibility tests, exclusion of drugs inappropriate for clinical translation, and dose dependency tests, berberine, mubritinib, and pioglitazone were identified as a candidate. An in vitro BBSCB functional test revealed that berberine and mubritinib, but not pioglitazone, protected BBSCB from oxygen-glucose deprivation and reoxygenation stress. Additionally, these two drugs minimized BBSCB breakdown 1 day after cervical SCI in mice. Furthermore, berberine and mubritinib reduced neuronal loss and improved gait performance 8 weeks after SCI. Collectively, the current study established a useful HTSA to identify potential neuroprotective drugs by maintaining BBSCB function and demonstrated the neuroprotective effect of berberine and mubritinib after SCI.
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Berberina , Fármacos Neuroprotetores , Traumatismos da Medula Espinal , Camundongos , Humanos , Animais , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Fármacos Neuroprotetores/metabolismo , Berberina/farmacologia , Berberina/uso terapêutico , Neuroproteção , Células Endoteliais , Ensaios de Triagem em Larga Escala , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Peróxido de Hidrogênio/uso terapêutico , Reprodutibilidade dos Testes , Traumatismos da Medula Espinal/tratamento farmacológico , Medula Espinal , Pioglitazona/metabolismo , Pioglitazona/farmacologia , Pioglitazona/uso terapêutico , Recuperação de Função FisiológicaRESUMO
Adult T-cell leukemia/lymphoma (ATLL) is one of the aggressive peripheral T-cell neoplasms with a poor prognosis. Accumulating evidence demonstrates that escape from adaptive immunity is a hallmark of ATLL pathogenesis. However, the mechanisms by which ATLL cells evade natural killer (NK)-cell-mediated immunity have been poorly understood. Here we show that CD48 expression in ATLL cells determines the sensitivity for NK-cell-mediated cytotoxicity against ATLL cells. We performed unbiased genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screening using 2 ATLL-derived cell lines and discovered CD48 as one of the best-enriched genes whose knockout conferred resistance to YT1-NK cell line-mediated cytotoxicity. The ability of CD48-knockout ATLL cells to evade NK-cell effector function was confirmed using human primary NK cells with reduced interferon-γ (IFNγ) induction and degranulation. We found that primary ATLL cells had reduced CD48 expression along with disease progression. Furthermore, other subgroups among aggressive peripheral T-cell lymphomas (PTCLs) also expressed lower concentrations of CD48 than normal T cells, suggesting that CD48 is a key molecule in malignant T-cell evasion of NK-cell surveillance. Thus, this study demonstrates that CD48 expression is likely critical for malignant T-cell lymphoma cell regulation of NK-cell-mediated immunity and provides a rationale for future evaluation of CD48 as a molecular biomarker in NK-cell-associated immunotherapies.
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Leucemia-Linfoma de Células T do Adulto , Linfoma de Células T Periférico , Adulto , Humanos , Antígeno CD48/genética , Antígeno CD48/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Leucemia-Linfoma de Células T do Adulto/genética , Linfoma de Células T Periférico/genética , Células Matadoras NaturaisRESUMO
MS is a powerful methodology for chemical screening to directly quantify substrates and products of enzymes, but its low throughput has been an issue. Recently, an acoustic liquid-handling apparatus (Echo®) used for rapid nano-dispensing has been coupled to a high-sensitivity mass spectrometer to create the Echo® MS system, and we applied this system to screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3CL protease inhibitors. Primary screening of 32033 chemical samples was completed in 12 h. Among the hits showing selective, dose-dependent 3CL-inhibitory activity, 8 compounds showed antiviral activity in cell-based assay.
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Tratamento Farmacológico da COVID-19 , Inibidores de Proteases , Acústica , Ensaios de Triagem em Larga Escala/métodos , Humanos , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , SARS-CoV-2RESUMO
To develop methodology to predict the potential druggability of middle molecules, we examined the structure, solubility, and permeability relationships of a diverse library (HKDL ver.1) consisting of 510 molecules (359 natural product derivatives, 76 non-natural products, 46 natural products, and 29 non-natural product derivatives). The library included peptides, depsipeptides, macrolides, and lignans, and 476 of the 510 compounds had a molecular weight in the range of 500-2000 Da. The solubility and passive diffusion velocity of the middle molecules were assessed using the parallel artificial membrane permeability assay (PAMPA). Quantitative values of solubility of 471 molecules and passive diffusion velocity of 287 molecules were obtained, and their correlations with the structural features of the molecules were examined. Based on the results, we propose a method to predict the passive diffusion characteristics of middle molecules from their three-dimensional structural features.
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Bibliotecas de Moléculas Pequenas/química , Difusão , Membranas Artificiais , Estrutura Molecular , Permeabilidade , SolubilidadeRESUMO
Adult T-cell leukemia (ATL) is a CD4+ T-cell neoplasm caused by human T-cell leukemia virus type I. As the prognosis for patients with ATL remains extremely poor due to resistance to conventional chemotherapy regimens, introduction of novel therapeutic agents is needed. Previous studies have reported that nucleosides 2'-deoxy-2'-methylidenecytidine (DMDC) and its derivative 2'-deoxy-2'-methylidene-5-fluorocytidine (FDMDC) exhibit antitumor activities in T-cell acute lymphoblastic leukemia (T-ALL) and solid tumor cell lines. Another nucleoside, 1-(2-azido-2-deoxy-ß-D-arabinofuranosyl)cytosine (cytarazid), is considered a therapeutic drug with antitumor activity in human solid tumors. In this study, we investigated the effects of these nucleosides on cell growth in vitro and in vivo using relevant leukemia cell lines and NOD/Shi-scid, IL-2Rgnull (NOG) mice, respectively. The nucleosides demonstrated significant cytotoxic effects in ATL and T-ALL cell lines. Intraperitoneal administration of FDMDC and DMDC into tumor-bearing NOG mice resulted in significant suppression of tumor growth without lethal side effects. Our findings support a therapeutic application of these nucleosides against tumor progression by targeting DNA polymerase-dependent DNA synthesis in patients with ATL.
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The Notch receptor serves a fundamental role in the regulation of cell fate determination through intracellular signal transmission. Mutation of the Notch receptor results in abnormal active signaling, leading to the development of diseases involving abnormal cell growth, including malignant tumors. Therefore, the Notch signaling pathway is a useful pharmacological target for the treatment of cancer. In the present study, a compound screening system was designed to identify inhibitors of the Notch signaling targeting Notch intracellular domain (NICD). A total of 9,600 compounds were analyzed using the Michigan Cancer Foundation7 (MCF7) human breast adenocarcinoma cell line and the SHSY5Y human neuroblastoma cell line with the reporter assay system using an artificial protein encoding a partial Notch carboxylterminal fragment fused to the Gal4 DNAbinding domain. The molecular mechanism underlying the inhibition of Notch signaling by a hit compound was further validated using biochemical and cell biological approaches. Using the screening system, a potential candidate, Notch signaling inhibitor1 (NSI1), was isolated which showed 50% inhibition at 6.1 µM in an exogenous Notch signaling system. In addition, NSI1 suppressed the nuclear translocation of NICD and endogenous gene expression of hairy and enhancer of split1, indicating that NSI1 specifically targets Notch. Notably, NSI1 suppressed the cell viability of MCF7 cells and another human breast adenocarcinoma cell line, MDAMB231 exhibiting constitutive and high Notch signaling activity, whereas no significant effect was observed in the SHSY5Y cells bearing a lower Notch signaling activity. NSI1 significantly suppressed the viability of SHSY5Y cells expressing exogenous human Notch1. These results indicate that NSI1 is a novel Notch signaling inhibitor and suggest its potential as a useful drug for the treatment of diseases induced by constitutively active Notch signaling.
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Domínios e Motivos de Interação entre Proteínas , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular , Sobrevivência Celular/efeitos dos fármacos , Descoberta de Drogas/métodos , Humanos , Ligação Proteica , Transporte Proteico , Receptores Notch/química , Receptores Notch/genéticaRESUMO
Inosine 5'-monophosphate dehydrogenase (IMPDH) is an essential enzyme for the production of guanine nucleotides. Disruption of IMPDH activity has been explored as a therapeutic strategy for numerous purposes, such as for anticancer, immunosuppression, antiviral, and antimicrobial therapy. In the present study, we established a luciferase-based high-throughput screening system to identify IMPDH inhibitors from our chemical library of known bioactive small molecules. The screening of 1400 compounds resulted in the discovery of three irreversible inhibitors: disulfiram, bronopol, and ebselen. Each compound has a distinct chemical moiety that differs from other reported IMPDH inhibitors. Further evaluation revealed that these compounds are potent inhibitors of IMPDHs with kon values of 0.7 × 104 to 9.3 × 104 M-1·s-1. Both disulfiram and bronopol exerted similar degree of inhibition to protozoan and mammalian IMPDHs. Ebselen showed an intriguing difference in mode of inhibition for different IMPDHs, with reversible and irreversible inhibition to each Cryptosporidium parvum IMPDH and human IMPDH type II, respectively. In the preliminary efficacy experiment against cryptosporidiosis in severe combined immunodeficiency (SCID) mouse, a decrease in the number of oocyst shed was observed upon the oral administration of disulfiram and bronopol, providing an early clinical proof-of-concept for further utilization of these compounds as IMPDH inhibitors.
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Descoberta de Drogas , Reposicionamento de Medicamentos , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala/métodos , IMP Desidrogenase/antagonistas & inibidores , Animais , Azóis/química , Azóis/isolamento & purificação , Azóis/farmacologia , Cryptosporidium parvum/enzimologia , Dissulfiram/química , Dissulfiram/isolamento & purificação , Dissulfiram/farmacologia , Inibidores Enzimáticos/química , Humanos , IMP Desidrogenase/metabolismo , Isoindóis , Cinética , Camundongos , Camundongos SCID , Compostos Organosselênicos/química , Compostos Organosselênicos/isolamento & purificação , Compostos Organosselênicos/farmacologia , Estudo de Prova de Conceito , Propilenoglicóis/química , Propilenoglicóis/isolamento & purificação , Propilenoglicóis/farmacologia , Bibliotecas de Moléculas PequenasRESUMO
AU-rich elements (ARE) exist in the 3'-untranslated regions of the mRNA transcribed from cell growth-related genes such as proto-oncogenes, cyclin-related genes, and growth factors. HuR binds and stabilizes ARE-mRNA. HuR is expressed abundantly in cancer cells and related malignant phenotypes. HuR knockdown attenuates the malignant phenotype of oral cancer cells. In this study, we screened 1570 compounds in the approved drug library by differential scanning fluorimetry (DSF) to discover a HuR-targeted compound. Firstly, 55 compounds were selected by DSF. Then, 8 compounds that showed a shift in the melting temperature value in a concentration-dependent manner were selected by DSF. Of them, suramin, an anti-trypanosomal drug, binds to HuR, exhibiting fast-on and fast-off kinetic behavior on surface plasmon resonance (SPR). We confirmed that suramin significantly decreased mRNA and protein expression of cyclin A2 and cyclin B1. The cyclin A2 and cyclin B1 mRNAs were destabilized by suramin. Furthermore, the motile and invasive activities of a tongue carcinoma cell line treated with suramin were markedly lower than those of control cells. The above findings suggest that suramin binds to HuR and inhibits its function. We also showed that the anticancer effects of suramin were caused by the inhibition of HuR function, indicating its potential as a novel therapeutic agent in the treatment of oral cancer. Our results suggest that suramin, via its different mechanism, may effectively suppress progressive oral cancer that cannot be controlled using other anticancer agents.
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Antineoplásicos/farmacologia , Proteína Semelhante a ELAV 1/metabolismo , Suramina/farmacologia , Neoplasias da Língua/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Proteína Semelhante a ELAV 1/genética , Humanos , Bibliotecas de Moléculas Pequenas , Neoplasias da Língua/tratamento farmacológico , Cicatrização/efeitos dos fármacosRESUMO
Rabies remains an invariably fatal neurological disease despite the availability of a preventive vaccination and post-exposure prophylaxis that must be immediately administered to the exposed individual before symptom onset. There is no effective medication for treatment during the symptomatic phase. Ribavirin, a guanine nucleoside analog, is a potent inhibitor of rabies virus (RABV) replication in vitro but lacks clinical efficacy. Therefore, we attempted to identify potential ribavirin analogs with comparable or superior anti-RABV activity. Antiviral activity and cytotoxicity of the compounds were initially examined in human neuroblastoma cells. Among the tested compounds, two exhibited a 5- to 27-fold higher anti-RABV activity than ribavirin. Examination of the anti-RABV mechanisms of action of the compounds using time-of-addition and minigenome assays revealed that they inhibited viral genome replication and transcription. Addition of exogenous guanosine to RABV-infected cells diminished the antiviral activity of the compounds, suggesting that they are involved in guanosine triphosphate (GTP) pool depletion by inhibiting inosine monophosphate dehydrogenase (IMPDH). Taken together, our findings underline the potency of nucleoside analogs as a class of antiviral compounds for the development of novel agents against RABV.
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Antivirais/farmacologia , Nucleosídeos/farmacologia , Vírus da Raiva/efeitos dos fármacos , Ribavirina/farmacologia , Animais , Linhagem Celular , Descoberta de Drogas , Humanos , Camundongos , Raiva/tratamento farmacológico , Raiva/prevenção & controle , Ribavirina/química , Replicação Viral/efeitos dos fármacosRESUMO
We accomplished divergent synthesis of potent kinase inhibitor BAY 61-3606 (1) and 27 derivatives via conjugation of imidazo[1,2-c]pyrimidine and indole ring compounds with aromatic (including pyridine) derivatives by means of palladium-catalyzed cross-coupling reaction. Spleen tyrosine kinase (Syk) and germinal center kinase (Gck, MAP4K2) inhibition assays showed that some of the synthesized compounds were selective Gck inhibitors.
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Imidazóis/química , Inibidores de Proteínas Quinases/síntese química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirimidinas/química , Catálise , Avaliação Pré-Clínica de Medicamentos , Quinases do Centro Germinativo , Humanos , Imidazóis/síntese química , Imidazóis/metabolismo , Indóis/química , Concentração Inibidora 50 , Niacinamida/análogos & derivados , Niacinamida/química , Niacinamida/metabolismo , Paládio/química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Pirimidinas/síntese química , Pirimidinas/metabolismo , Relação Estrutura-Atividade , Quinase Syk/antagonistas & inibidores , Quinase Syk/metabolismoRESUMO
Glioblastoma (GBM), one of the most malignant human cancers, frequently recurs despite multimodal treatment with surgery and chemo/radiotherapies. GBM-initiating cells (GICs) are the likely cell-of-origin in recurrences, as they proliferate indefinitely, form tumors in vivo, and are resistant to chemo/radiotherapies. It is therefore crucial to find chemicals that specifically kill GICs. We established temozolomide (the standard medicine for GBM)-resistant GICs (GICRs) and used the cells for chemical screening. Here, we identified 1-(3-C-ethynyl-ß-d-ribopentofuranosyl) uracil (EUrd) as a selective drug for targeting GICRs. EUrd induced the death in GICRs more effectively than their parental GICs, while it was less toxic to normal neural stem cells. We demonstrate that the cytotoxic effect of EUrd on GICRs partly depended on the increased expression of uridine-cytidine kinase-like 1 (UCKL1) and the decreased one of 5'-nucleotidase cytosolic III (NT5C3), which regulate uridine-monophosphate synthesis positively and negatively respectively. Together, these findings suggest that EUrd can be used as a new therapeutic drug for GBM with the expression of surrogate markers UCKL1 and NT5C3. Stem Cells 2016;34:2016-2025.