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1.
Mol Cells ; 44(9): 688-695, 2021 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-34518443

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has become a global health concern. Various SARS-CoV-2 vaccines have been developed and are being used for vaccination worldwide. However, no therapeutic agents against coronavirus disease 2019 (COVID-19) have been developed so far; therefore, new therapeutic agents are urgently needed. In the present study, we evaluated several hepatitis C virus direct-acting antivirals as potential candidates for drug repurposing against COVID-19. Theses include asunaprevir (a protease inhibitor), daclatasvir (an NS5A inhibitor), and sofosbuvir (an RNA polymerase inhibitor). We found that asunaprevir, but not sofosbuvir and daclatasvir, markedly inhibited SARS-CoV-2-induced cytopathic effects in Vero E6 cells. Both RNA and protein levels of SARS-CoV-2 were significantly decreased by treatment with asunaprevir. Moreover, asunaprevir profoundly decreased virion release from SARS-CoV-2-infected cells. A pseudoparticle entry assay revealed that asunaprevir blocked SARS-CoV-2 infection at the binding step of the viral life cycle. Furthermore, asunaprevir inhibited SARS-CoV-2 propagation in human lung Calu-3 cells. Collectively, we found that asunaprevir displays broad-spectrum antiviral activity and therefore might be worth developing as a new drug repurposing candidate for COVID-19.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Isoquinolinas/farmacologia , SARS-CoV-2/crescimento & desenvolvimento , Sulfonamidas/farmacologia , Inibidores de Protease Viral/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Carbamatos/farmacologia , Linhagem Celular , Chlorocebus aethiops , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Células HEK293 , Hepacivirus/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Pirrolidinas/farmacologia , SARS-CoV-2/efeitos dos fármacos , Sofosbuvir/farmacologia , Valina/análogos & derivados , Valina/farmacologia , Células Vero , Proteínas não Estruturais Virais/antagonistas & inibidores
2.
Nat Commun ; 12(1): 5523, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34535646

RESUMO

RNA polymerase inhibition plays an important role in the regulation of transcription in response to environmental changes and in the virus-host relationship. Here we present the high-resolution structures of two such RNAP-inhibitor complexes that provide the structural bases underlying RNAP inhibition in archaea. The Acidianus two-tailed virus encodes the RIP factor that binds inside the DNA-binding channel of RNAP, inhibiting transcription by occlusion of binding sites for nucleic acid and the transcription initiation factor TFB. Infection with the Sulfolobus Turreted Icosahedral Virus induces the expression of the host factor TFS4, which binds in the RNAP funnel similarly to eukaryotic transcript cleavage factors. However, TFS4 allosterically induces a widening of the DNA-binding channel which disrupts trigger loop and bridge helix motifs. Importantly, the conformational changes induced by TFS4 are closely related to inactivated states of RNAP in other domains of life indicating a deep evolutionary conservation of allosteric RNAP inhibition.


Assuntos
RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/química , Vírus/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Proteínas Arqueais/metabolismo , Microscopia Crioeletrônica , DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína , Fatores de Tempo , Proteínas Virais/metabolismo , Viroides/metabolismo
3.
Nucleic Acids Res ; 49(15): 8777-8784, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34365509

RESUMO

Transcribing RNA polymerase (RNAP) can fall into backtracking, phenomenon when the 3' end of the transcript disengages from the template DNA. Backtracking is caused by sequences of the nucleic acids or by misincorporation of erroneous nucleotides. To resume productive elongation backtracked complexes have to be resolved through hydrolysis of RNA. There is currently no consensus on the mechanism of catalysis of this reaction by Escherichia coli RNAP. Here we used Salinamide A, that we found inhibits RNAP catalytic domain Trigger Loop (TL), to show that the TL is required for RNA cleavage during proofreading of misincorporation events but plays little role during cleavage in sequence-dependent backtracked complexes. Results reveal that backtracking caused by misincorporation is distinct from sequence-dependent backtracking, resulting in different conformations of the 3' end of RNA within the active center. We show that the TL is required to transfer the 3' end of misincorporated transcript from cleavage-inefficient 'misincorporation site' into the cleavage-efficient 'backtracked site', where hydrolysis takes place via transcript-assisted catalysis and is largely independent of the TL. These findings resolve the controversy surrounding mechanism of RNA hydrolysis by E. coli RNA polymerase and indicate that the TL role in RNA cleavage has diverged among bacteria.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , RNA Mensageiro/metabolismo , Elongação da Transcrição Genética , Domínio Catalítico , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/química , Depsipeptídeos/química , Depsipeptídeos/farmacologia , Escherichia coli/enzimologia , Escherichia coli/genética , Hidrólise , Clivagem do RNA
4.
Sci Rep ; 11(1): 1029, 2021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33441878

RESUMO

Tuberculosis (TB) is an infectious disease caused by the bacillus Mycobacterium tuberculosis (Mtb). The present work reports the design and synthesis of a hybrid of the precursors of rifampicin and clofazimine, which led to the discovery of a novel Rifaphenazine (RPZ) molecule with potent anti-TB activity. In addition, the efficacy of RPZ was evaluated in-vitro using the reference strain Mtb H37Rv. Herein, 2,3 diamino phenazine, a precursor of an anti-TB drug clofazimine, was tethered to the rifampicin core. This 2,3 diamino phenazine did not have an inherent anti-TB activity even at a concentration of up to 2 µg/mL, while rifampicin did not exhibit any activity against Mtb at a concentration of 0.1 µg/mL. However, the synthesized novel Rifaphenzine (RPZ) inhibited 78% of the Mtb colonies at a drug concentration of 0.1 µg/mL, while 93% of the bacterial colonies were killed at 0.5 µg/mL of the drug. Furthermore, the Minimum Inhibitory Concentration (MIC) value for RPZ was 1 µg/mL. Time-kill studies revealed that all bacterial colonies were killed within a period of 24 h. The synthesized novel molecule was characterized using high-resolution mass spectroscopy and NMR spectroscopy. Cytotoxicity studies (IC50) were performed on human monocytic cell line THP-1, and the determined IC50 value was 96 µg/mL, which is non-cytotoxic.


Assuntos
Antituberculosos/síntese química , Clofazimina/análogos & derivados , Mycobacterium tuberculosis/efeitos dos fármacos , Rifampina/análogos & derivados , Antituberculosos/química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Clofazimina/síntese química , Clofazimina/química , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/química , Desenho de Fármacos , Descoberta de Drogas , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Monócitos/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Rifampina/síntese química , Rifampina/química , Células THP-1
5.
Angew Chem Int Ed Engl ; 60(15): 8164-8173, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33476096

RESUMO

Nucleosidic and oligonucleotidic diarylethenes (DAEs) are an emerging class of photochromes with high application potential. However, their further development is hampered by the poor understanding of how the chemical structure modulates the photochromic properties. Here we synthesized 26 systematically varied deoxyuridine- and deoxycytidine-derived DAEs and analyzed reaction quantum yields, composition of the photostationary states, thermal and photochemical stability, and reversibility. This analysis identified two high-performance photoswitches with near-quantitative, fully reversible back-and-forth switching and no detectable thermal or photochemical deterioration. When incorporated into an oligonucleotide with the sequence of a promotor, the nucleotides maintained their photochromism and allowed the modulation of the transcription activity of T7 RNA polymerase with an up to 2.4-fold turn-off factor, demonstrating the potential for optochemical control of biological processes.


Assuntos
RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Desenvolvimento de Medicamentos , Inibidores Enzimáticos/farmacologia , Etilenos/farmacologia , Oligonucleotídeos/farmacologia , Nucleosídeos de Pirimidina/farmacologia , Proteínas Virais/antagonistas & inibidores , Bacteriófago T7/enzimologia , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Etilenos/síntese química , Etilenos/química , Oligonucleotídeos/síntese química , Oligonucleotídeos/química , Processos Fotoquímicos , Nucleosídeos de Pirimidina/síntese química , Nucleosídeos de Pirimidina/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
6.
Nat Rev Microbiol ; 19(2): 95-109, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33122819

RESUMO

Transcription of DNA is a fundamental process in all cellular organisms. The enzyme responsible for transcription, RNA polymerase, is conserved in general architecture and catalytic function across the three domains of life. Diverse mechanisms are used among and within the different branches to regulate transcription initiation. Mechanistic studies of transcription initiation in bacteria are especially amenable because the promoter recognition and melting steps are much less complicated than in eukaryotes or archaea. Also, bacteria have critical roles in human health as pathogens and commensals, and the bacterial RNA polymerase is a proven target for antibiotics. Recent biophysical studies of RNA polymerases and their inhibition, as well as transcription initiation and transcription factors, have detailed the mechanisms of transcription initiation in phylogenetically diverse bacteria, inspiring this Review to examine unifying and diverse themes in this process.


Assuntos
Archaea/genética , Bactérias/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Iniciação da Transcrição Genética/fisiologia , Antibacterianos/farmacologia , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Regiões Promotoras Genéticas/genética , RNA Bacteriano/genética , Fator sigma/metabolismo
7.
Sci Rep ; 10(1): 21309, 2020 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-33277558

RESUMO

Multidrug-resistant Mycobacterium tuberculosis (MDR-TB) accounts for 3.7% of new cases of TB annually worldwide and is a major threat to global public health. Due to the prevalence of the MDR-TB and extensively drug resistant tuberculosis (XDR-TB) cases, there is an urgent need for new drugs with novel mechanisms of action. CarD, a global transcription regulator in MTB, binds RNAP and activates transcription by stabilizing the transcription initiation open-promoter complex (RPo). CarD is required for MTB viability and it has highly conserved homologues in many eubacteria. A fluorescence polarization (FP) assay which monitors the association of MTB RNAP, native rRNA promoter DNA and CarD has been developed. Overall, our objective is to identify and characterize small molecule inhibitors which block the CarD/RNAP interaction and to understand the mechanisms by which CarD interacts with the molecules. We expect that the development of a new and improved anti-TB compound with a novel mechanism of action will relieve the burden of resistance. This CarD FP assay is amenable to HTS and is an enabling tool for future novel therapeutic discovery.


Assuntos
RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Polarização de Fluorescência , Mycobacterium tuberculosis/enzimologia , RNA Polimerases Dirigidas por DNA/metabolismo , Ensaios de Triagem em Larga Escala
8.
Eur J Pharmacol ; 887: 173568, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32956644

RESUMO

In December 2019, an unprecedented outbreak of pneumonia associated with a novel coronavirus disease 2019 (COVID-19) emerged in Wuhan City, Hubei province, China. The virus that caused the disease was officially named by the World Health Organization (WHO) as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). According to the high transmission rate of SARS-CoV-2, it became a global pandemic and public health emergency within few months. Since SARS-CoV-2 is genetically 80% homologous with the SARS-CoVs family, it is hypothesized that medications developed for the treatment of SARS-CoVs may be useful in the control and management of SARS-CoV-2. In this regard, some medication being tested in clinical trials and in vitro studies include anti-viral RNA polymerase inhibitors, HIV-protease inhibitors, anti-inflammatory agents, angiotensin converting enzyme type 2 (ACE 2) blockers, and some other novel medications. In this communication, we reviewed the general characteristics of medications, medical usage, mechanism of action, as well as SARS-CoV-2 related trials.


Assuntos
Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/fisiopatologia , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/fisiopatologia , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Antivirais/farmacologia , Antivirais/uso terapêutico , COVID-19 , Cloroquina/análogos & derivados , Cloroquina/uso terapêutico , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Humanos , Pandemias
9.
Virol J ; 17(1): 141, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32972430

RESUMO

BACKGROUND: The COVID-19 causing coronavirus is an enveloped RNA virus that utilizes an enzyme RNA dependent RNA polymerase for its replication. Favipiravir (FVP) triphosphate, a purine nucleoside analog, inhibits that enzyme. We have conducted this systematic review and meta-analysis on efficacy and safety of the drug FVP as a treatment for COVID-19. METHODS: Databases like Pubmed, Pubmed Central, Scopus, Embase, Google Scholar, preprint sites, and clinicaltirals.gov were searched. The studies with the standard of care (SOC) and FVP as a treatment drug were considered as the treatment group and the SOC with other antivirals and supportive care as the control group. Quantitative synthesis was done using RevMan 5.4. Clinical improvement, negative conversion of reverse transcription-polymerase chain reaction (RT-PCR), adverse effects, and oxygen requirements were studied. RESULTS: We identified a total of 1798 studies after searching the electronic databases. Nine in the qualitative studies and four studies in the quantitative synthesis met the criteria. There was a significant clinical improvement in the FVP group on the 14th day compared to the control group (RR 1.29, 1.08-1.54). Clinical deterioration rates were less likely in the FVP group though statistically not significant (OR 0.59, 95% CI 0.30-1.14) at the endpoint of study (7-15 days). The meta-analysis showed no significant differences between the two groups on viral clearance (day 14: RR 1.06, 95% CI 0.84-1.33), non-invasive ventilation or oxygen requirement (OR 0.76, 95% CI 0.42-1.39), and adverse effects (OR 0.69, 0.13-3.57). There are 31 randomized controlled trials (RCTs) registered in different parts of the world focusing FVP for COVID-19 treatment. CONCLUSION: There is a significant clinical and radiological improvement following treatment with FVP in comparison to the standard of care with no significant differences on viral clearance, oxygen support requirement and side effect profiles.


Assuntos
Amidas/uso terapêutico , Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Pirazinas/uso terapêutico , Amidas/efeitos adversos , Antivirais/efeitos adversos , Betacoronavirus/enzimologia , COVID-19 , Ensaios Clínicos Fase II como Assunto , Ensaios Clínicos Fase III como Assunto , Infecções por Coronavirus/virologia , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Bases de Dados Factuais , Inibidores Enzimáticos/uso terapêutico , Humanos , Pandemias , Pneumonia Viral/virologia , Pirazinas/efeitos adversos , Ensaios Clínicos Controlados Aleatórios como Assunto , SARS-CoV-2 , Padrão de Cuidado , Resultado do Tratamento
10.
J Med Chem ; 63(18): 10380-10395, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32816483

RESUMO

Chronic hepatitis C (CHC) is a major liver disease caused by the hepatitis C virus. The current standard of care for CHC can achieve cure rates above 95%; however, the drugs in current use are administered for a period of 8-16 weeks. A combination of safe and effective drugs with a shorter treatment period is highly desirable. We report synthesis and biological evaluation of a series of 2',3'- and 2',4'-substituted guanosine nucleotide analogues. Their triphosphates exhibited potent inhibition of the HCV NS5B polymerase with IC50 as low as 0.13 µM. In the HCV replicon assay, the phosphoramidate prodrugs of these analogues demonstrated excellent activity with EC50 values as low as 5 nM. A lead compound AL-611 showed high levels of the nucleoside 5'-triphosphate in vitro in primary human hepatocytes and in vivo in dog liver following oral administration.


Assuntos
Antivirais/farmacologia , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Nucleotídeos de Guanina/farmacologia , Hepacivirus/efeitos dos fármacos , Pró-Fármacos/farmacologia , Animais , Antivirais/síntese química , Antivirais/toxicidade , Cães , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/toxicidade , Feminino , Nucleotídeos de Guanina/síntese química , Nucleotídeos de Guanina/toxicidade , Humanos , Masculino , Pró-Fármacos/síntese química , Pró-Fármacos/toxicidade , Proteínas não Estruturais Virais/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos
12.
J Biol Chem ; 295(39): 13432-13443, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32737197

RESUMO

Enzyme kinetic analysis reveals a dynamic relationship between enzymes and their substrates. Overall enzyme activity can be controlled by both protein expression and various cellular regulatory systems. Interestingly, the availability and concentrations of intracellular substrates can constantly change, depending on conditions and cell types. Here, we review previously reported enzyme kinetic parameters of cellular and viral DNA and RNA polymerases with respect to cellular levels of their nucleotide substrates. This broad perspective exposes a remarkable co-evolution scenario of DNA polymerase enzyme kinetics with dNTP levels that can vastly change, depending on cell proliferation profiles. Similarly, RNA polymerases display much higher Km values than DNA polymerases, possibly due to millimolar range rNTP concentrations found in cells (compared with micromolar range dNTP levels). Polymerases are commonly targeted by nucleotide analog inhibitors for the treatments of various human diseases, such as cancers and viral pathogens. Because these inhibitors compete against natural cellular nucleotides, the efficacy of each inhibitor can be affected by varying cellular nucleotide levels in their target cells. Overall, both kinetic discrepancy between DNA and RNA polymerases and cellular concentration discrepancy between dNTPs and rNTPs present pharmacological and mechanistic considerations for therapeutic discovery.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Nucleotídeos/metabolismo , Animais , RNA Polimerases Dirigidas por DNA/metabolismo , Humanos , Cinética , Especificidade por Substrato
13.
Nucleic Acids Res ; 48(14): 7914-7923, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32652039

RESUMO

Bacterial RNA polymerase is a potent target for antibiotics, which utilize a plethora of different modes of action, some of which are still not fully understood. Ureidothiophene (Urd) was found in a screen of a library of chemical compounds for ability to inhibit bacterial transcription. The mechanism of Urd action is not known. Here, we show that Urd inhibits transcription at the early stage of closed complex formation by blocking interaction of RNA polymerase with the promoter -10 element, while not affecting interactions with -35 element or steps of transcription after promoter closed complex formation. We show that mutation in the region 1.2 of initiation factor σ decreases sensitivity to Urd. The results suggest that Urd may directly target σ region 1.2, which allosterically controls the recognition of -10 element by σ region 2. Alternatively, Urd may block conformational changes of the holoenzyme required for engagement with -10 promoter element, although by a mechanism distinct from that of antibiotic fidaxomycin (lipiarmycin). The results suggest a new mode of transcription inhibition involving the regulatory domain of σ subunit, and potentially pinpoint a novel target for development of new antibacterials.


Assuntos
Antibacterianos/farmacologia , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Regiões Promotoras Genéticas , Tiofenos/farmacologia , Iniciação da Transcrição Genética/efeitos dos fármacos , Antibacterianos/química , Bactérias/enzimologia , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fator sigma/antagonistas & inibidores , Fator sigma/química , Tiofenos/química
14.
Nat Microbiol ; 5(10): 1232-1246, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32661315

RESUMO

Paramyxoviruses such as human parainfluenza virus type-3 (HPIV3) and measles virus (MeV) are a substantial health threat. In a high-throughput screen for inhibitors of HPIV3 (a major cause of acute respiratory infection), we identified GHP-88309-a non-nucleoside inhibitor of viral polymerase activity that possesses unusual broad-spectrum activity against diverse paramyxoviruses including respiroviruses (that is, HPIV1 and HPIV3) and morbilliviruses (that is, MeV). Resistance profiles of distinct target viruses overlapped spatially, revealing a conserved binding site in the central cavity of the viral polymerase (L) protein that was validated by photoaffinity labelling-based target mapping. Mechanistic characterization through viral RNA profiling and in vitro MeV polymerase assays identified a block in the initiation phase of the viral polymerase. GHP-88309 showed nanomolar potency against HPIV3 isolates in well-differentiated human airway organoid cultures, was well tolerated (selectivity index > 7,111) and orally bioavailable, and provided complete protection against lethal infection in a Sendai virus mouse surrogate model of human HPIV3 disease when administered therapeutically 48 h after infection. Recoverees had acquired robust immunoprotection against reinfection, and viral resistance coincided with severe attenuation. This study provides proof of the feasibility of a well-behaved broad-spectrum allosteric antiviral and describes a chemotype with high therapeutic potential that addresses major obstacles of anti-paramyxovirus drug development.


Assuntos
Antivirais/química , Antivirais/farmacologia , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Respirovirus/efeitos dos fármacos , Respirovirus/enzimologia , Imunidade Adaptativa , Administração Oral , Regulação Alostérica , Animais , Antivirais/administração & dosagem , Linhagem Celular , Inibidores Enzimáticos/administração & dosagem , Humanos , Imuno-Histoquímica , Camundongos , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Ligação Proteica , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Mucosa Respiratória/virologia , Respirovirus/imunologia , Relação Estrutura-Atividade
16.
J Gen Virol ; 101(4): 385-398, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32553055

RESUMO

The influenza A virus (IAV) ribonucleoprotein (vRNP) complex consists of polymerase subunits, nucleoprotein (NP) and viral RNA and is responsible for RNA transcription and replication. Interactions between the vRNP complex and host factors play important roles in virus replication, pathogenicity and species tropism. In this study, Strep-tag affinity purification coupled with mass spectrometry was used to identify host factors that interact with IAV vRNP complex in infected human cells. We purified vRNP complex from HEK 293T cells infected with a recombinant mouse-adapted IAV (A/Chicken/Hubei/489/2004) containing a Strep-tag PB2 subunit and identified Y-box-binding protein 3 (YBX3) as a negative regulator of IAV replication. Overexpression of YBX3 inhibited the virus replication, viral protein expression and vRNA synthesis. Conversely, RNAi knockdown of YBX3 resulted in significantly increased virus growth rate. Furthermore, knockdown of YBX3 augmented the nuclear accumulation of NP and viral primary transcription in infected cells. Our results suggest that YBX3 restricts IAV replication by interacting with vRNP complex and subsequently imparing its function.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas de Choque Térmico/metabolismo , Vírus da Influenza A/genética , Vírus da Influenza A/metabolismo , Proteínas Nucleares/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas do Core Viral/metabolismo , Replicação Viral , Células A549 , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Núcleo Celular/metabolismo , Citoplasma/metabolismo , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Cães , Células HEK293 , Proteínas de Choque Térmico/genética , Interações entre Hospedeiro e Microrganismos , Humanos , Vírus da Influenza A/enzimologia , Vírus da Influenza A/crescimento & desenvolvimento , Células Madin Darby de Rim Canino , Espectrometria de Massas , Camundongos , Ligação Proteica , RNA Interferente Pequeno , RNA Viral/metabolismo , Transcrição Genética , Regulação para Cima , Proteínas do Core Viral/genética , Replicação Viral/fisiologia
17.
Biochemistry (Mosc) ; 85(5): 523-530, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32571182

RESUMO

In the pathogenesis of the infectious process in the respiratory tract by SARS, MERS, and COVID-19 coronaviruses, two stages can be distinguished: early (etiotropic) and late (pathogenetic) ones. In the first stage, when the virus multiplication and accumulation are prevalent under insufficient host immune response, the use of chemotherapeutic agents blocking the reproduction of the virus is reasonable to suppress the development of the disease. This article considers six major chemotherapeutic classes aimed at certain viral targets: inhibitors of viral RNA polymerase, inhibitors of viral protease Mpro, inhibitors of proteolytic activation of viral protein S allowing virus entry into the target cell, inhibitors of virus uncoating in cellular endosomes, compounds of exogenous interferons, and compounds of natural and recombinant virus-neutralizing antibodies. In the second stage, when the multiplication of the virus decreases and threatening pathological processes of excessive inflammation, acute respiratory distress syndrome, pulmonary edema, hypoxia, and secondary bacterial pneumonia and sepsis events develop, a pathogenetic therapeutic approach including extracorporeal blood oxygenation, detoxification, and anti-inflammatory and anti-bacterial therapy seems to be the most effective way for the patient's recovery.


Assuntos
Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Terapia de Alvo Molecular/métodos , Pneumonia Viral/tratamento farmacológico , Anticorpos Antivirais/uso terapêutico , Antivirais/farmacologia , Betacoronavirus/enzimologia , Betacoronavirus/imunologia , COVID-19 , Proteases 3C de Coronavírus , Infecções por Coronavirus/virologia , Cisteína Endopeptidases , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Quimioterapia Combinada , Humanos , Interferon alfa-2/uso terapêutico , Pandemias , Pneumonia Viral/virologia , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
18.
Nucleic Acids Res ; 48(11): e64, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32352514

RESUMO

The ability to block gene expression in bacteria with the catalytically inactive mutant of Cas9, known as dCas9, is quickly becoming a standard methodology to probe gene function, perform high-throughput screens, and engineer cells for desired purposes. Yet, we still lack a good understanding of the design rules that determine on-target activity for dCas9. Taking advantage of high-throughput screening data, we fit a model to predict the ability of dCas9 to block the RNA polymerase based on the target sequence, and validate its performance on independently generated datasets. We further design a novel genome wide guide RNA library for E. coli MG1655, EcoWG1, using our model to choose guides with high activity while avoiding guides which might be toxic or have off-target effects. A screen performed using the EcoWG1 library during growth in rich medium improved upon previously published screens, demonstrating that very good performances can be attained using only a small number of well designed guides. Being able to design effective, smaller libraries will help make CRISPRi screens even easier to perform and more cost-effective. Our model and materials are available to the community through crispr.pasteur.fr and Addgene.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Escherichia coli/genética , Ensaios de Triagem em Larga Escala , RNA Guia/genética , Sequência de Bases , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Conjuntos de Dados como Assunto , Modelos Lineares , Reprodutibilidade dos Testes
19.
Chem Biol Drug Des ; 96(2): 812-824, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32259386

RESUMO

A new approach is introduced for the construction of a predictive quantitative structure-activity relationship model in which only ligand-receptor (LR) interaction features are used as relevant descriptors. This approach combines the benefit of the random forest (RF) as a new variable selection method with the intrinsic capability of the artificial neural network (ANN). The interaction information of the ligand-receptor (LR) complex was used as molecular docking descriptors. The most relevant descriptors were selected using the RF technique and used as inputs of ANN. The proposed RF ANN (RF-LM-ANN) method was optimized and then evaluated by the prediction of pEC50 for some of the azine derivatives as non-nucleoside reverse transcriptase inhibitors. RF-LM-ANN model under the optimal conditions was evaluated using internal (validation) and external test sets. The determination coefficients of the external test and validation sets were 0.88 and 0.89, respectively. The mean square deviation (MSE) values for the prediction of biological activities in the external test and validation sets were found to be 0.10 and 0.11, respectively. The results obtained demonstrated the good prediction ability and high generalizability of the proposed RF-LM-ANN model based on the MMDs alone.


Assuntos
RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Inibidores Enzimáticos/química , Compostos Heterocíclicos/química , Ligantes , Simulação de Acoplamento Molecular , Redes Neurais de Computação , Ligação Proteica , Relação Quantitativa Estrutura-Atividade
20.
Bioorg Med Chem Lett ; 30(11): 127146, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32249118

RESUMO

Antibiotic resistance in bacteria has been an emerging public health problem, thus discovery of novel and effective antibiotics is urgent. A series of novel hybrids of N-aryl pyrrothine-base α-pyrone hybrids was designed, synthesized and evaluated as bacterial RNA polymerase (RNAP) inhibitors. Among them, compound 13c exhibited potent antibacterial activity against antibiotic-resistant S. aureus with the minimum inhibitory concentration (MIC) in the range of 1-4 µg/mL. Moreover, compound 13c exhibited strong inhibitory activity against E.coli RNAP with IC50 value of 16.06 µM, and cytotoxicity in HepG2 cells with IC50 value of 7.04 µM. The molecular docking study further suggested that compound 13c binds to the switch region of bacterial RNAP. In summary, compound 13c is a novel bacterial RNAP inhibitor, and a promising lead compound for further optimization.


Assuntos
Antibacterianos/síntese química , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/química , Escherichia coli/enzimologia , Pironas/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Sítios de Ligação , Candida albicans/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , RNA Polimerases Dirigidas por DNA/metabolismo , Inibidores Enzimáticos/metabolismo , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Células Hep G2 , Humanos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Pironas/metabolismo , Pironas/farmacologia , Relação Estrutura-Atividade
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