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1.
Nat Commun ; 12(1): 5156, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34526504

RESUMO

R-loops are by-products of transcription that must be tightly regulated to maintain genomic stability and gene expression. Here, we describe a mechanism for the regulation of the R-loop-specific helicase, senataxin (SETX), and identify the ubiquitin specific peptidase 11 (USP11) as an R-loop regulator. USP11 de-ubiquitinates SETX and its depletion increases SETX K48-ubiquitination and protein turnover. Loss of USP11 decreases SETX steady-state levels and reduces R-loop dissolution. Ageing of USP11 knockout cells restores SETX levels via compensatory transcriptional downregulation of the E3 ubiquitin ligase, KEAP1. Loss of USP11 reduces SETX enrichment at KEAP1 promoter, leading to R-loop accumulation, enrichment of the endonuclease XPF and formation of double-strand breaks. Overexpression of KEAP1 increases SETX K48-ubiquitination, promotes its degradation and R-loop accumulation. These data define a ubiquitination-dependent mechanism for SETX regulation, which is controlled by the opposing activities of USP11 and KEAP1 with broad applications for cancer and neurological disease.


Assuntos
DNA Helicases/genética , DNA/genética , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Enzimas Multifuncionais/genética , Processamento de Proteína Pós-Traducional , Proteostase/genética , RNA Helicases/genética , Tioléster Hidrolases/genética , Linhagem Celular , Senescência Celular/genética , DNA/química , DNA/metabolismo , DNA Helicases/antagonistas & inibidores , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Células HEK293 , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/antagonistas & inibidores , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Enzimas Multifuncionais/antagonistas & inibidores , Enzimas Multifuncionais/metabolismo , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estabilidade Proteica , Proteólise , RNA Helicases/antagonistas & inibidores , RNA Helicases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Tioléster Hidrolases/antagonistas & inibidores , Tioléster Hidrolases/metabolismo , Ubiquitinação
2.
Int J Biol Macromol ; 190: 636-648, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34517025

RESUMO

SARS-CoV-2 nucleocapsid (N) protein undergoes RNA-induced phase separation (LLPS) and sequesters the host key stress granule (SG) proteins, Ras-GTPase-activating protein SH3-domain-binding protein 1 and 2 (G3BP1 and G3BP2) to inhibit SG formation. This will allow viral packaging and propagation in host cells. Based on a genomic-guided meta-analysis, here we identify upstream regulatory elements modulating the expression of G3BP1 and G3BP2 (collectively called G3BP1/2). Using this strategy, we have identified FOXA1, YY1, SYK, E2F-1, and TGFBR2 as activators and SIN3A, SRF, and AKT-1 as repressors of G3BP1/2 genes. Panels of the activators and repressors were then used to identify drugs that change their gene expression signatures. Two drugs, imatinib, and decitabine have been identified as putative modulators of G3BP1/2 genes and their regulators, suggesting their role as COVID-19 mitigation agents. Molecular docking analysis suggests that both drugs bind to G3BP1/2 with a much higher affinity than the SARS-CoV-2 N protein. This study reports imatinib and decitabine as candidate drugs against N protein and G3BP1/2 protein.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , COVID-19/tratamento farmacológico , Proteínas do Nucleocapsídeo de Coronavírus/química , DNA Helicases/química , Decitabina/química , Mesilato de Imatinib/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas de Ligação a Poli-ADP-Ribose/química , RNA Helicases/química , Proteínas com Motivo de Reconhecimento de RNA/química , Proteínas de Ligação a RNA/química , SARS-CoV-2/química , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , COVID-19/metabolismo , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , DNA Helicases/antagonistas & inibidores , DNA Helicases/metabolismo , Decitabina/farmacologia , Sistemas de Liberação de Medicamentos , Genômica , Mesilato de Imatinib/farmacologia , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/antagonistas & inibidores , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/antagonistas & inibidores , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismo , SARS-CoV-2/metabolismo
3.
Nat Commun ; 12(1): 4848, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381037

RESUMO

There is currently a lack of effective drugs to treat people infected with SARS-CoV-2, the cause of the global COVID-19 pandemic. The SARS-CoV-2 Non-structural protein 13 (NSP13) has been identified as a target for anti-virals due to its high sequence conservation and essential role in viral replication. Structural analysis reveals two "druggable" pockets on NSP13 that are among the most conserved sites in the entire SARS-CoV-2 proteome. Here we present crystal structures of SARS-CoV-2 NSP13 solved in the APO form and in the presence of both phosphate and a non-hydrolysable ATP analog. Comparisons of these structures reveal details of conformational changes that provide insights into the helicase mechanism and possible modes of inhibition. To identify starting points for drug development we have performed a crystallographic fragment screen against NSP13. The screen reveals 65 fragment hits across 52 datasets opening the way to structure guided development of novel antiviral agents.


Assuntos
Metiltransferases/química , RNA Helicases/química , SARS-CoV-2/química , Proteínas não Estruturais Virais/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Apoenzimas/química , Apoenzimas/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Modelos Moleculares , Fosfatos/química , Fosfatos/metabolismo , Conformação Proteica , RNA Helicases/antagonistas & inibidores , RNA Helicases/metabolismo , RNA Viral/química , RNA Viral/metabolismo , SARS-CoV-2/enzimologia , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo
4.
Molecules ; 26(13)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206406

RESUMO

Spanish flu, polio epidemics, and the ongoing COVID-19 pandemic are the most profound examples of severe widespread diseases caused by RNA viruses. The coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands affordable and reliable assays for testing antivirals. To test inhibitors of viral proteases, we have developed an inexpensive high-throughput assay based on fluorescent energy transfer (FRET). We assayed an array of inhibitors for papain-like protease from SARS-CoV-2 and validated it on protease from the tick-borne encephalitis virus to emphasize its versatility. The reaction progress is monitored as loss of FRET signal of the substrate. This robust and reproducible assay can be used for testing the inhibitors in 96- or 384-well plates.


Assuntos
Antivirais/farmacologia , Transferência Ressonante de Energia de Fluorescência/métodos , Ensaios de Triagem em Larga Escala/métodos , Inibidores de Proteases/farmacologia , Vírus de RNA/enzimologia , COVID-19/tratamento farmacológico , Proteases Semelhantes à Papaína de Coronavírus/antagonistas & inibidores , Proteases Semelhantes à Papaína de Coronavírus/química , Proteases Semelhantes à Papaína de Coronavírus/genética , Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Avaliação Pré-Clínica de Medicamentos , Vírus da Encefalite Transmitidos por Carrapatos/enzimologia , Corantes Fluorescentes/química , Humanos , RNA Helicases/antagonistas & inibidores , RNA Helicases/química , RNA Helicases/genética , RNA Helicases/metabolismo , SARS-CoV-2/enzimologia , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
5.
Biochem J ; 478(13): 2405-2423, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34198322

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global public health challenge. While the efficacy of vaccines against emerging and future virus variants remains unclear, there is a need for therapeutics. Repurposing existing drugs represents a promising and potentially rapid opportunity to find novel antivirals against SARS-CoV-2. The virus encodes at least nine enzymatic activities that are potential drug targets. Here, we have expressed, purified and developed enzymatic assays for SARS-CoV-2 nsp13 helicase, a viral replication protein that is essential for the coronavirus life cycle. We screened a custom chemical library of over 5000 previously characterized pharmaceuticals for nsp13 inhibitors using a fluorescence resonance energy transfer-based high-throughput screening approach. From this, we have identified FPA-124 and several suramin-related compounds as novel inhibitors of nsp13 helicase activity in vitro. We describe the efficacy of these drugs using assays we developed to monitor SARS-CoV-2 growth in Vero E6 cells.


Assuntos
Antivirais/química , Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos , RNA Helicases/antagonistas & inibidores , SARS-CoV-2/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Chlorocebus aethiops , Ensaios Enzimáticos , Transferência Ressonante de Energia de Fluorescência , Ensaios de Triagem em Larga Escala , RNA Helicases/metabolismo , Reprodutibilidade dos Testes , SARS-CoV-2/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Suramina/farmacologia , Células Vero , Proteínas não Estruturais Virais/metabolismo
6.
J Immunol ; 206(10): 2453-2467, 2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33941659

RESUMO

The detection of intracellular nucleic acids is a fundamental mechanism of host defense against infections. The dysregulated nucleic acid sensing, however, is a major cause for a number of autoimmune diseases. In this study, we report that GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) is critical for both intracellular DNA- and RNA-induced immune responses. We found that in both human and mouse cells, the deletion of G3BP1 led to the dampened cGAS activation by DNA and the insufficient binding of RNA by RIG-I. We further found that resveratrol (RSVL), a natural compound found in grape skin, suppressed both intracellular DNA- and RNA-induced type I IFN production through inhibiting G3BP1. Importantly, using experimental mouse models for Aicardi-Goutières syndrome, an autoimmune disorder found in humans, we demonstrated that RSVL effectively alleviated intracellular nucleic acid-stimulated autoimmune responses. Thus, our study demonstrated a broader role of G3BP1 in sensing different kinds of intracellular nucleic acids and presented RSVL as a potential treatment for autoimmune conditions caused by dysregulated nucleic acid sensing.


Assuntos
Autoimunidade/genética , DNA Helicases/deficiência , DNA Helicases/metabolismo , Espaço Intracelular/metabolismo , Ácidos Nucleicos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/deficiência , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/deficiência , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/deficiência , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Transdução de Sinais/genética , Células A549 , Animais , Autoimunidade/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , DNA Helicases/antagonistas & inibidores , DNA Helicases/genética , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Espaço Intracelular/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Helicases/antagonistas & inibidores , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/antagonistas & inibidores , Proteínas com Motivo de Reconhecimento de RNA/genética , Resveratrol/administração & dosagem , Transdução de Sinais/imunologia , Transfecção
7.
Sci Rep ; 11(1): 10290, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33986405

RESUMO

As the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic engulfs millions worldwide, the quest for vaccines or drugs against the virus continues. The helicase protein of SARS-CoV-2 represents an attractive target for drug discovery since inhibition of helicase activity can suppress viral replication. Using in silico approaches, we have identified drugs that interact with SARS-CoV-2 helicase based on the presence of amino acid arrangements matching binding sites of drugs in previously annotated protein structures. The drugs exhibiting an RMSD of ≤ 3.0 Å were further analyzed using molecular docking, molecular dynamics (MD) simulation, and post-MD analyses. Using these approaches, we found 12 drugs that showed strong interactions with SARS-CoV-2 helicase amino acids. The analyses were performed using the recently available SARS-CoV-2 helicase structure (PDB ID: 5RL6). Based on the MM-GBSA approach, out of the 12 drugs, two drugs, namely posaconazole and grazoprevir, showed the most favorable binding energy, - 54.8 and - 49.1 kcal/mol, respectively. Furthermore, of the amino acids found conserved among all human coronaviruses, 10/11 and 10/12 were targeted by, respectively, grazoprevir and posaconazole. These residues are part of the crucial DEAD-like helicase C and DEXXQc_Upf1-like/ DEAD-like helicase domains. Strong interactions of posaconazole and grazoprevir with conserved amino acids indicate that the drugs can be potent against SARS-CoV-2. Since the amino acids are conserved among the human coronaviruses, the virus is unlikely to develop resistance mutations against these drugs. Since these drugs are already in use, they may be immediately repurposed for SARS-CoV-2 therapy.


Assuntos
Amidas/farmacologia , Carbamatos/farmacologia , Ciclopropanos/farmacologia , Reposicionamento de Medicamentos , Inibidores Enzimáticos/farmacologia , Quinoxalinas/farmacologia , RNA Helicases/antagonistas & inibidores , SARS-CoV-2/enzimologia , Sulfonamidas/farmacologia , Triazóis/farmacologia , Antivirais/farmacologia , COVID-19/tratamento farmacológico , Reposicionamento de Medicamentos/métodos , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Domínios Proteicos/efeitos dos fármacos , RNA Helicases/química , RNA Helicases/metabolismo , SARS-CoV-2/efeitos dos fármacos , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/química , Proteínas Virais/metabolismo
8.
ACS Appl Mater Interfaces ; 13(22): 25715-25726, 2021 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-34036784

RESUMO

Dengue virus (DENV), an arbovirus transmitted by mosquitoes, causes infectious diseases such as dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. Despite the dangers posed by DENV, there are no approved antiviral drugs for treatment of DENV infection. Considering the potential for a global dengue outbreak, rapid development of antiviral agents against DENV infections is crucial as a preemptive measure; thus, the selection of apparent drug targets, such as the viral enzymes involved in the viral life cycle, is recommended. Helicase, a potential drug target in DENV, is a crucial viral enzyme that unwinds double-stranded viral RNA, releasing single-stranded RNA genomes during viral replication. Therefore, an inhibitor of helicase activity could serve as a direct-acting antiviral agent. Here, we introduce an RNA helicase assay based on graphene oxide, which enables fluorescence-based analysis of RNA substrate-specific helicase enzyme activity. This assay demonstrated high reliability and ability for high-throughput screening, identifying a new helicase inhibitor candidate, micafungin (MCFG), from an FDA-approved drug library. As a direct-acting antiviral agent targeting RNA helicase, MCFG inhibits DENV proliferation in cells and an animal model. Notably, in vivo, MCFG treatment reduced viremia, inflammatory cytokine levels, and viral loads in several tissues and improved survival rates by up to 40% in a lethal mouse model. Therefore, we suggest MCFG as a potential direct-acting antiviral drug candidate.


Assuntos
Antivirais/farmacologia , Técnicas Biossensoriais/métodos , Vírus da Dengue/efeitos dos fármacos , Dengue/tratamento farmacológico , Grafite/química , Micafungina/farmacologia , RNA Helicases/antagonistas & inibidores , Animais , Antifúngicos/farmacologia , Antivirais/química , Dengue/enzimologia , Dengue/virologia , Vírus da Dengue/enzimologia , Ensaios de Triagem em Larga Escala/métodos , Camundongos , Nanopartículas/química , Replicação Viral
9.
Molecules ; 26(5)2021 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-33800013

RESUMO

With the emergence and global spread of the COVID-19 pandemic, the scientific community worldwide has focused on search for new therapeutic strategies against this disease. One such critical approach is targeting proteins such as helicases that regulate most of the SARS-CoV-2 RNA metabolism. The purpose of the current study was to predict a library of phytochemicals derived from diverse plant families with high binding affinity to SARS-CoV-2 helicase (Nsp13) enzyme. High throughput virtual screening of the Medicinal Plant Database for Drug Design (MPD3) database was performed on SARS-CoV-2 helicase using AutoDock Vina. Nilotinib, with a docking value of -9.6 kcal/mol, was chosen as a reference molecule. A compound (PubChem CID: 110143421, ZINC database ID: ZINC257223845, eMolecules: 43290531) was screened as the best binder (binding energy of -10.2 kcal/mol on average) to the enzyme by using repeated docking runs in the screening process. On inspection, the compound was disclosed to show different binding sites of the triangular pockets collectively formed by Rec1A, Rec2A, and 1B domains and a stalk domain at the base. The molecule is often bound to the ATP binding site (referred to as binding site 2) of the helicase enzyme. The compound was further discovered to fulfill drug-likeness and lead-likeness criteria, have good physicochemical and pharmacokinetics properties, and to be non-toxic. Molecular dynamic simulation analysis of the control/lead compound complexes demonstrated the formation of stable complexes with good intermolecular binding affinity. Lastly, affirmation of the docking simulation studies was accomplished by estimating the binding free energy by MMPB/GBSA technique. Taken together, these findings present further in silco investigation of plant-derived lead compounds to effectively address COVID-19.


Assuntos
Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , RNA Helicases/antagonistas & inibidores , RNA Helicases/metabolismo , SARS-CoV-2/enzimologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacocinética , Antivirais/toxicidade , Sítios de Ligação , Disponibilidade Biológica , COVID-19/tratamento farmacológico , Biologia Computacional/métodos , Bases de Dados de Compostos Químicos , Desenho de Fármacos , Humanos , Ligação de Hidrogênio , Metiltransferases/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Compostos Fitoquímicos/química , Compostos Fitoquímicos/metabolismo , Plantas Medicinais/química , Ligação Proteica , Domínios Proteicos/efeitos dos fármacos , Pirimidinas/química , Pirimidinas/metabolismo , Pirimidinas/farmacocinética , Pirimidinas/toxicidade , RNA Helicases/química , Relação Estrutura-Atividade , Termodinâmica , Proteínas não Estruturais Virais/química
10.
J Virol ; 95(12)2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-33789997

RESUMO

Enteroviruses belong to the genus Enterovirus of the family Picornaviridae and include four human enterovirus groups (EV-A to -D): the epidemic of enteroviruses such as human enterovirus A71 (EV-A71) and coxsackievirus A16 (CVA16) is a threat to global public health. Enteroviral protein 2C is the most conserved nonstructural protein among all enteroviruses and possesses RNA helicase activity that plays pivotal roles during enteroviral life cycles, which makes 2C an attractive target for developing antienterovirus drugs. In this study, we designed a peptide, named 2CL, based on the structure of EV-A71 2C. This peptide effectively impaired the oligomerization of EV-A71 2C protein and inhibited the RNA helicase activities of 2C proteins encoded by EV-A71 and CVA16, both of which belong to EV-A, and showed potent antiviral efficacy against EV-A71 and CVA16 in cells. Moreover, the 2CL treatment elicited a strong in vivo protective efficacy against lethal EV-A71 challenge. In addition, the antiviral strategy of targeting the 2C helicase activity can be applied to inhibit the replication of EV-B. Either 2CL or B-2CL, the peptide redesigned based on the 2CL-corresponding sequence of EV-Bs, could exert effective antiviral activity against two important EV-Bs, coxsackievirus B3 and echovirus 11. Together, our findings demonstrated that targeting the helicase activity of 2C with a rationally designed peptide is an efficient antiviral strategy against enteroviruses, and 2CL and B-2CL show promising clinical potential to be further developed as broad-spectrum antienterovirus drugs.IMPORTANCE Enteroviruses are a large group of positive-sense single-stranded RNA viruses and include numerous human pathogens, such as enterovirus A71 (EV-A71), coxsackieviruses, and echoviruses. However, no approved EV antiviral drugs are available. Enteroviral 2C is the most conserved nonstructural protein among all enteroviruses and contains the RNA helicase activity critical for the viral life cycle. Herein, according to the structure of EV-A71 2C, we designed a peptide that effectively inhibited the RNA helicase activities of EV-A71- and coxsackievirus A16 (CVA16)-encoded 2C proteins. Moreover, this peptide exerted potent antiviral effects against EV-A71 and CVA16 in cells and elicited therapeutic efficacy against lethal EV-A71 challenge in vivo Furthermore, we demonstrate that the strategy of targeting the 2C helicase activity can be used for other relevant enteroviruses, including coxsackievirus B3 and echovirus 11. In summary, our findings provide compelling evidence that the designed peptides targeting the helicase activity of 2C could be broad-spectrum antivirals for enteroviruses.


Assuntos
Antivirais/farmacologia , Proteínas de Transporte/antagonistas & inibidores , Enterovirus Humano A/efeitos dos fármacos , Infecções por Enterovirus/tratamento farmacológico , Peptídeos/farmacologia , RNA Helicases/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Antivirais/química , Antivirais/uso terapêutico , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Linhagem Celular , Desenho de Fármacos , Enterovirus Humano A/química , Enterovirus Humano A/fisiologia , Enterovirus Humano B/efeitos dos fármacos , Enterovirus Humano B/fisiologia , Infecções por Enterovirus/virologia , Humanos , Camundongos , Camundongos Endogâmicos ICR , Peptídeos/química , Peptídeos/uso terapêutico , RNA Helicases/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
11.
PLoS One ; 16(2): e0246181, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33596235

RESUMO

The 2019 emergence of, SARS-CoV-2 has tragically taken an immense toll on human life and far reaching impacts on society. There is a need to identify effective antivirals with diverse mechanisms of action in order to accelerate preclinical development. This study focused on five of the most established drug target proteins for direct acting small molecule antivirals: Nsp5 Main Protease, Nsp12 RNA-dependent RNA polymerase, Nsp13 Helicase, Nsp16 2'-O methyltransferase and the S2 subunit of the Spike protein. A workflow of solvent mapping and free energy calculations was used to identify and characterize favorable small-molecule binding sites for an aromatic pharmacophore (benzene). After identifying the most favorable sites, calculated ligand efficiencies were compared utilizing computational fragment screening. The most favorable sites overall were located on Nsp12 and Nsp16, whereas the most favorable sites for Nsp13 and S2 Spike had comparatively lower ligand efficiencies relative to Nsp12 and Nsp16. Utilizing fragment screening on numerous possible sites on Nsp13 helicase, we identified a favorable allosteric site on the N-terminal zinc binding domain (ZBD) that may be amenable to virtual or biophysical fragment screening efforts. Recent structural studies of the Nsp12:Nsp13 replication-transcription complex experimentally corroborates ligand binding at this site, which is revealed to be a functional Nsp8:Nsp13 protein-protein interaction site in the complex. Detailed structural analysis of Nsp13 ZBD conformations show the role of induced-fit flexibility in this ligand binding site and identify which conformational states are associated with efficient ligand binding. We hope that this map of over 200 possible small-molecule binding sites for these drug targets may be of use for ongoing discovery, design, and drug repurposing efforts. This information may be used to prioritize screening efforts or aid in the process of deciphering how a screening hit may bind to a specific target protein.


Assuntos
Antivirais/farmacologia , COVID-19/virologia , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Metiltransferases/metabolismo , RNA Helicases/metabolismo , SARS-CoV-2/efeitos dos fármacos , Proteínas não Estruturais Virais/metabolismo , Sítio Alostérico , Sítios de Ligação , COVID-19/tratamento farmacológico , COVID-19/metabolismo , Biologia Computacional/métodos , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/metabolismo , RNA-Polimerase RNA-Dependente de Coronavírus/antagonistas & inibidores , RNA-Polimerase RNA-Dependente de Coronavírus/química , Humanos , Metiltransferases/antagonistas & inibidores , Metiltransferases/química , Modelos Moleculares , Terapia de Alvo Molecular , Ligação Proteica , RNA Helicases/antagonistas & inibidores , RNA Helicases/química , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Replicação Viral/efeitos dos fármacos
12.
Expert Opin Ther Pat ; 31(4): 339-350, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33593200

RESUMO

Introduction: Coronaviruses encode a helicase that is essential for viral replication and represents an excellent antiviral target. However, only a few coronavirus helicase inhibitors have been patented. These patents include drug-like compound SSYA10-001, aryl diketo acids (ADK), and dihydroxychromones. Additionally, adamantane-derived bananins, natural flavonoids, one acrylamide derivative [(E)-3-(furan-2-yl)-N-(4-sulfamoylphenyl)acrylamide], a purine derivative (7-ethyl-8-mercapto-3-methyl-3,7-dihydro-1 H-purine-2,6-dione), and a few bismuth complexes. The IC50 of patented inhibitors ranges between 0.82 µM and 8.95 µM, depending upon the assays used. Considering the urgency of clinical interventions against Coronavirus Disease-19 (COVID-19), it is important to consider developing antiviral portfolios consisting of small molecules.Areas covered: This review examines coronavirus helicases as antiviral targets, and the potential of previously patented and experimental compounds to inhibit the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) helicase.Expert opinion: Small molecule coronavirus helicase inhibitors represent attractive pharmacological modalities for the treatment of coronaviruses such as SARS-CoV and SARS-CoV-2. Rightfully so, the current emphasis is focused upon the development of vaccines. However, vaccines may not work for everyone and broad-based adoption of vaccinations is an increasingly challenging societal endeavor. Therefore, it is important to develop additional pharmacological antivirals against the highly conserved coronavirus helicases to broadly protect against this and subsequent coronavirus epidemics.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Desenvolvimento de Medicamentos , Metiltransferases/antagonistas & inibidores , RNA Helicases/antagonistas & inibidores , SARS-CoV-2/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Humanos , Metiltransferases/química , Metiltransferases/fisiologia , Patentes como Assunto , RNA Helicases/química , RNA Helicases/fisiologia , Triazóis/farmacologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/fisiologia
13.
Mol Cell ; 81(5): 1027-1042.e4, 2021 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-33453166

RESUMO

Alternative lengthening of telomeres (ALT) is mediated by break-induced replication (BIR), but how BIR is regulated at telomeres is poorly understood. Here, we show that telomeric BIR is a self-perpetuating process. By tethering PML-IV to telomeres, we induced telomere clustering in ALT-associated PML bodies (APBs) and a POLD3-dependent ATR response at telomeres, showing that BIR generates replication stress. Ablation of BLM helicase activity in APBs abolishes telomere synthesis but causes multiple chromosome bridges between telomeres, revealing a function of BLM in processing inter-telomere BIR intermediates. Interestingly, the accumulation of BLM in APBs requires its own helicase activity and POLD3, suggesting that BIR triggers a feedforward loop to further recruit BLM. Enhancing BIR induces PIAS4-mediated TRF2 SUMOylation, and PIAS4 loss deprives APBs of repair proteins and compromises ALT telomere synthesis. Thus, a BLM-driven and PIAS4-mediated feedforward loop operates in APBs to perpetuate BIR, providing a critical mechanism to extend ALT telomeres.


Assuntos
Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Retroalimentação Fisiológica , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas Inibidoras de STAT Ativados/genética , RNA Helicases/genética , Homeostase do Telômero , Telômero/química , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/antagonistas & inibidores , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Corpos de Inclusão Intranuclear/genética , Corpos de Inclusão Intranuclear/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas Inibidoras de STAT Ativados/antagonistas & inibidores , Proteínas Inibidoras de STAT Ativados/metabolismo , RNA Helicases/antagonistas & inibidores , RNA Helicases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , RecQ Helicases/genética , RecQ Helicases/metabolismo , Transdução de Sinais , Sumoilação , Telômero/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/genética
14.
Methods Mol Biol ; 2209: 35-52, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33201461

RESUMO

RNA helicases are proteins found in all kingdoms of life, and they are associated with all processes involving RNA from transcription to decay. They use NTP binding and hydrolysis to unwind duplexes, to remodel RNA structures and protein-RNA complexes, and to facilitate the unidirectional metabolism of biological processes. Viral, bacterial, and eukaryotic parasites have an intimate need for RNA helicases in their reproduction. Moreover, various disorders, like cancers, are often associated with a perturbation of the host's helicase activity. Thus, RNA helicases provide a rich source of targets for the development of therapeutic or prophylactic drugs. In this review, we provide an overview of the different targeting strategies against helicases, the different types of compounds explored, the proposed inhibitory mechanisms of the compounds on the proteins, and the therapeutic potential of these compounds in the treatment of various disorders.


Assuntos
Inibidores Enzimáticos , RNA Helicases , Animais , Infecções Bacterianas/tratamento farmacológico , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Humanos , Infecções por Protozoários/tratamento farmacológico , RNA Helicases/antagonistas & inibidores , RNA Helicases/fisiologia , Viroses/tratamento farmacológico
15.
Methods Mol Biol ; 2209: 53-72, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33201462

RESUMO

RNA helicases are responsible for virtually all of RNA metabolism. Viral and bacterial pathogens typically encode their own RNA helicases. Hence, this family of enzymes is increasingly recognized as potential targets for treatment of a variety of diseases. However, the conserved structural similarities among helicase families present an obstacle to the idea of developing specific inhibitors. In order to identify potential modulators of RNA helicase activity, rapid screening approaches are needed. This has been accomplished by optimizing and adapting standard helicase assays to function in high-throughput modalities. These optimized assays have enabled the application of rapid screening approaches to be applied toward discovering helicase inhibitors. This chapter provides detailed protocols for utilizing a medium to high-throughput approach for inhibitor discovery.


Assuntos
Ensaios Enzimáticos/métodos , Inibidores Enzimáticos/análise , RNA Helicases/antagonistas & inibidores , RNA/química , Fluorescência , Humanos
16.
Nat Commun ; 11(1): 4979, 2020 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-33020468

RESUMO

Cellular senescence is a known driver of carcinogenesis and age-related diseases, yet senescence is required for various physiological processes. However, the mechanisms and factors that control the negative effects of senescence while retaining its benefits are still elusive. Here, we show that the rasGAP SH3-binding protein 1 (G3BP1) is required for the activation of the senescent-associated secretory phenotype (SASP). During senescence, G3BP1 achieves this effect by promoting the association of the cyclic GMP-AMP synthase (cGAS) with cytosolic chromatin fragments. In turn, G3BP1, through cGAS, activates the NF-κB and STAT3 pathways, promoting SASP expression and secretion. G3BP1 depletion or pharmacological inhibition impairs the cGAS-pathway preventing the expression of SASP factors without affecting cell commitment to senescence. These SASPless senescent cells impair senescence-mediated growth of cancer cells in vitro and tumor growth in vivo. Our data reveal that G3BP1 is required for SASP expression and that SASP secretion is a primary mediator of senescence-associated tumor growth.


Assuntos
Senescência Celular/fisiologia , DNA Helicases/metabolismo , Neoplasias/patologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Células A549 , Animais , Carcinogênese , Linhagem Celular , Movimento Celular , Citocinas/metabolismo , DNA Helicases/antagonistas & inibidores , DNA Helicases/deficiência , Humanos , Inflamação , Camundongos , Neoplasias/metabolismo , Nucleotidiltransferases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Proteínas de Ligação a Poli-ADP-Ribose/deficiência , RNA Helicases/antagonistas & inibidores , RNA Helicases/deficiência , Proteínas com Motivo de Reconhecimento de RNA/antagonistas & inibidores , Proteínas com Motivo de Reconhecimento de RNA/deficiência , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Fator de Transcrição RelA/metabolismo
17.
J Phys Chem Lett ; 11(21): 9144-9151, 2020 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-33052685

RESUMO

The raging COVID-19 pandemic caused by SARS-CoV-2 has infected tens of millions of people and killed several hundred thousand patients worldwide. Currently, there are no effective drugs or vaccines available for treating coronavirus infections. In this study, we have focused on the SARS-CoV-2 helicase (Nsp13), which is critical for viral replication and the most conserved nonstructural protein within the coronavirus family. Using homology modeling that couples published electron-density with molecular dynamics (MD)-based structural refinements, we generated structural models of the SARS-CoV-2 helicase in its apo- and ATP/RNA-bound conformations. We performed virtual screening of ∼970 000 chemical compounds against the ATP-binding site to identify potential inhibitors. Herein, we report docking hits of approved human drugs targeting the ATP-binding site. Importantly, two of our top drug hits have significant activity in inhibiting purified recombinant SARS-CoV-2 helicase, providing hope that these drugs can be potentially repurposed for the treatment of COVID-19.


Assuntos
Antivirais/química , Betacoronavirus/enzimologia , RNA Helicases/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Antivirais/metabolismo , Antivirais/uso terapêutico , Betacoronavirus/isolamento & purificação , Sítios de Ligação , COVID-19 , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Estrutura Terciária de Proteína , RNA Helicases/química , RNA Helicases/metabolismo , SARS-CoV-2 , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo
18.
Int J Biol Macromol ; 163: 1687-1696, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32980406

RESUMO

SARS-CoV-2 has caused COVID-19 outbreak with nearly 2 M infected people and over 100K death worldwide, until middle of April 2020. There is no confirmed drug for the treatment of COVID-19 yet. As the disease spread fast and threaten human life, repositioning of FDA approved drugs may provide fast options for treatment. In this aspect, structure-based drug design could be applied as a powerful approach in distinguishing the viral drug target regions from the host. Evaluation of variations in SARS-CoV-2 genome may ease finding specific drug targets in the viral genome. In this study, 3458 SARS-CoV-2 genome sequences isolated from all around the world were analyzed. Incidence of C17747T and A17858G mutations were observed to be much higher than others and they were on Nsp13, a vital enzyme of SARS-CoV-2. Effect of these mutations was evaluated on protein-drug interactions using in silico methods. The most potent drugs were found to interact with the key and neighbor residues of the active site responsible from ATP hydrolysis. As result, cangrelor, fludarabine, folic acid and polydatin were determined to be the most potent drugs which have potency to inhibit both the wild type and mutant SARS-CoV-2 helicase. Clinical data supporting these findings would be important towards overcoming COVID-19.


Assuntos
Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Metiltransferases/antagonistas & inibidores , Pneumonia Viral/tratamento farmacológico , RNA Helicases/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Betacoronavirus/enzimologia , Betacoronavirus/genética , Sítios de Ligação , COVID-19 , Simulação por Computador , Infecções por Coronavirus/virologia , Aprovação de Drogas , Reposicionamento de Medicamentos , Ácido Fólico/farmacologia , Genoma Viral , Glucosídeos/farmacologia , Humanos , Metiltransferases/química , Metiltransferases/genética , Metiltransferases/metabolismo , Simulação de Acoplamento Molecular , Mutação , Pandemias , Pneumonia Viral/virologia , RNA Helicases/química , RNA Helicases/genética , RNA Helicases/metabolismo , SARS-CoV-2 , Estilbenos/farmacologia , Vidarabina/análogos & derivados , Vidarabina/farmacologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
19.
Mini Rev Med Chem ; 20(18): 1900-1907, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32767936

RESUMO

The global spread of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that causes COVID-19 has become a source of grave medical and socioeconomic concern to human society. Since its first appearance in the Wuhan region of China in December 2019, the most effective measures of managing the spread of SARS-CoV-2 infection have been social distancing and lockdown of human activity; the level of which has not been seen in our generations. Effective control of the viral infection and COVID-19 will ultimately depend on the development of either a vaccine or therapeutic agents. This article highlights the progresses made so far in these strategies by assessing key targets associated with the viral replication cycle. The key viral proteins and enzymes that could be targeted by new and repurposed drugs are discussed.


Assuntos
COVID-19/terapia , Proteases 3C de Coronavírus/antagonistas & inibidores , RNA Helicases/antagonistas & inibidores , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Anticorpos/uso terapêutico , Antiprotozoários/uso terapêutico , COVID-19/virologia , Proteases 3C de Coronavírus/metabolismo , Humanos , Nucleosídeos/análogos & derivados , Nucleosídeos/metabolismo , Nucleosídeos/uso terapêutico , Inibidores de Proteases/uso terapêutico , RNA Helicases/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/fisiologia
20.
Phys Chem Chem Phys ; 22(27): 15683-15695, 2020 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-32618974

RESUMO

The emergent Zika virus (ZIKV) infection has become a threat to global health due to its association with severe neurological abnormalities, namely Guillain-Barré Syndrome (GBS) in adults and Congenital Zika virus Syndrome (CZS) in neonates. Many studies are nowadays being conducted to find an effective antiviral drug against ZIKV. In particular, NS2B-NS3 protease is an attractive drug target due to its essential function in viral replication, although a drug is not yet commercially available. In this context, we present here a comparative structural study, based on quantum chemistry calculations, to analyze the intermolecular binding energies between the crystallographic structure of NS2B-NS3 protease and dipeptide boronic acid (cn-716) and aldehyde (acyl-KR-aldehyde) peptidomimetic inhibitors, by using the molecular fractionation with conjugate caps (MFCC) scheme within the density functional theory (DFT) formalism. Most intermolecular interactions in cn-716/NS2B-NS3 (acyl-KR-aldehyde/NS2B-NS3) are due to the amino acid residues Asp83*, His51, Asp129, Ser81*, Gly133, Ala132, Tyr161, Asn152 and Asp75 (Asp83*, Asp129, His51, Asn152, Tyr161, Tyr130, Gly153, Gly151, Asp75, Pro131, and Gly82). Additionally, we have considered missense mutation analysis of these residues to evaluate the destabilization and the increase of the flexibility of the protease, showing that mutation of the residues Tyr161 and Tyr130 causes more impact. Our simulations are a valuable tool for a better understanding of the binding mechanism of recognized inhibitors of NS2B-NS3 protease, and can lead to the rational design and development of novel anti-Zika drugs with improved efficiency.


Assuntos
Aldeídos/farmacologia , Antivirais/farmacologia , Ácidos Borônicos/farmacologia , Dipeptídeos/farmacologia , Inibidores de Proteases/farmacologia , Zika virus/efeitos dos fármacos , Aldeídos/química , Antivirais/química , Ácidos Borônicos/química , Teoria da Densidade Funcional , Dipeptídeos/química , Testes de Sensibilidade Microbiana , Conformação Molecular , Peptidomiméticos/antagonistas & inibidores , Peptidomiméticos/metabolismo , Inibidores de Proteases/química , RNA Helicases/antagonistas & inibidores , RNA Helicases/metabolismo , Serina Endopeptidases/metabolismo , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Zika virus/metabolismo
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