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1.
Eur Rev Med Pharmacol Sci ; 25(10): 3923-3932, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-34109607

RESUMO

Angiotensin converting enzyme 2 (ACE2) has potentially conflicting roles in health and disease. COVID-19 coronavirus binds to human cells via ACE2 receptor, which is expressed on almost all body organs. Boosting the ACE2 receptor levels on heart and lung cells may provide more cellular enter to virus thereby worsening the infection. Therefore, among the drug targets, ACE2 is suggested as a vital target of COVID-19 therapy. This hypothesis is based on the protective role of the drugs acting on ACE2. Therefore, this review discusses the impact and challenges of using ACE2 as a target in the current therapy of COVID-19.


Assuntos
Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Antivirais/química , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Monofosfato de Adenosina/uso terapêutico , Alanina/análogos & derivados , Alanina/química , Alanina/metabolismo , Alanina/uso terapêutico , Enzima de Conversão de Angiotensina 2/metabolismo , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/metabolismo , Anti-Inflamatórios não Esteroides/uso terapêutico , Antivirais/metabolismo , Antivirais/uso terapêutico , Azitromicina/química , Azitromicina/metabolismo , Azitromicina/uso terapêutico , COVID-19/tratamento farmacológico , COVID-19/virologia , Humanos , Hidroxicloroquina/química , Hidroxicloroquina/metabolismo , Hidroxicloroquina/uso terapêutico , SARS-CoV-2/isolamento & purificação , Vitamina D/química , Vitamina D/metabolismo , Vitamina D/uso terapêutico
2.
Molecules ; 26(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33947165

RESUMO

Dehydroalanine exists natively in certain proteins and can also be chemically made from the protein cysteine. As a strong Michael acceptor, dehydroalanine in proteins has been explored to undergo reactions with different thiolate reagents for making close analogues of post-translational modifications (PTMs), including a variety of lysine PTMs. The chemical reagent 2-nitro-5-thiocyanatobenzoic acid (NTCB) selectively modifies cysteine to form S-cyano-cysteine, in which the S-Cß bond is highly polarized. We explored the labile nature of this bond for triggering E2 elimination to generate dehydroalanine. Our results indicated that when cysteine is at the flexible C-terminal end of a protein, the dehydroalanine formation is highly effective. We produced ubiquitin and ubiquitin-like proteins with a C-terminal dehydroalanine residue with high yields. When cysteine is located at an internal region of a protein, the efficiency of the reaction varies with mainly hydrolysis products observed. Dehydroalanine in proteins such as ubiquitin and ubiquitin-like proteins can serve as probes for studying pathways involving ubiquitin and ubiquitin-like proteins and it is also a starting point to generate proteins with many PTM analogues; therefore, we believe that this NTCB-triggered dehydroalanine formation method will find broad applications in studying ubiquitin and ubiquitin-like protein pathways and the functional annotation of many PTMs in proteins such as histones.


Assuntos
Alanina/análogos & derivados , Cisteína/química , Proteínas/química , Tiocianatos/química , Alanina/química , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Modelos Moleculares , Conformação Proteica , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Recombinantes , Espectrometria de Massas por Ionização por Electrospray , Tiocianatos/farmacologia
3.
Int J Mol Sci ; 22(6)2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799639

RESUMO

Cas3 is a ssDNA-targeting nuclease-helicase essential for class 1 prokaryotic CRISPR immunity systems, which has been utilized for genome editing in human cells. Cas3-DNA crystal structures show that ssDNA follows a pathway from helicase domains into a HD-nuclease active site, requiring protein conformational flexibility during DNA translocation. In genetic studies, we had noted that the efficacy of Cas3 in CRISPR immunity was drastically reduced when temperature was increased from 30 °C to 37 °C, caused by an unknown mechanism. Here, using E. coli Cas3 proteins, we show that reduced nuclease activity at higher temperature corresponds with measurable changes in protein structure. This effect of temperature on Cas3 was alleviated by changing a single highly conserved tryptophan residue (Trp-406) into an alanine. This Cas3W406A protein is a hyperactive nuclease that functions independently from temperature and from the interference effector module Cascade. Trp-406 is situated at the interface of Cas3 HD and RecA1 domains that is important for maneuvering DNA into the nuclease active site. Molecular dynamics simulations based on the experimental data showed temperature-induced changes in positioning of Trp-406 that either blocked or cleared the ssDNA pathway. We propose that Trp-406 forms a 'gate' for controlling Cas3 nuclease activity via access of ssDNA to the nuclease active site. The effect of temperature in these experiments may indicate allosteric control of Cas3 nuclease activity caused by changes in protein conformations. The hyperactive Cas3W406A protein may offer improved Cas3-based genetic editing in human cells.


Assuntos
Proteínas Associadas a CRISPR/metabolismo , DNA Helicases/metabolismo , DNA de Cadeia Simples/metabolismo , DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Triptofano/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Alanina/química , Alanina/genética , Alanina/metabolismo , Sequência de Aminoácidos , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Domínio Catalítico/genética , Dicroísmo Circular , DNA/química , DNA/genética , DNA Helicases/química , DNA Helicases/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Edição de Genes/métodos , Humanos , Mutação de Sentido Incorreto , Conformação Proteica , Homologia de Sequência de Aminoácidos , Temperatura , Triptofano/química , Triptofano/genética
4.
Carbohydr Polym ; 264: 118011, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-33910715

RESUMO

Veklury™ by Gilead Sciences, Inc., containing antiviral drug, remdesivir (REM) has received emergency authorization in the USA and in Europe for COVID-19 therapy. Here, for the first time, we describe details of the non-covalent, host-guest type interaction between REM and the solubilizing excipient, sulfobutylether-beta-cyclodextrin (SBECD) that results in significant solubility enhancement. Complete amorphousness of the cyclodextrin-enabled REM formulation was demonstrated by X-ray diffraction, thermal analysis, Raman chemical mapping and electron microscopy/energy dispersive spectroscopy. The use of solubilizing carbohydrate resulted in a 300-fold improvement of the aqueous solubility of REM, and enhanced dissolution rate of the drug enabling the preparation of stable infusion solutions for therapy. 2D ROESY NMR spectroscopy provided information on the nature of REM-excipient interaction and indicated the presence of inclusion phenomenon and the electrostatic attraction between anionic SBECD and nitrogen-containing REM in aqueous solution.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Excipientes/química , beta-Ciclodextrinas/química , Monofosfato de Adenosina/química , Alanina/química , Antivirais/química , COVID-19/tratamento farmacológico , Varredura Diferencial de Calorimetria , Liofilização/métodos , Espectroscopia de Ressonância Magnética , Microscopia Eletrônica de Varredura , Simulação de Acoplamento Molecular , Nanofibras/química , Pós , Solubilidade , Análise Espectral Raman , Difração de Raios X
5.
Phys Chem Chem Phys ; 23(10): 5852-5863, 2021 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-33688867

RESUMO

COVID-19 has recently caused a global health crisis and an effective interventional therapy is urgently needed. Remdesivir is one effective inhibitor for SARS-CoV-2 viral RNA replication. It supersedes other NTP analogues because it not only terminates the polymerization activity of RNA-dependent RNA polymerase (RdRp), but also inhibits the proofreading activity of intrinsic exoribonuclease (ExoN). Even though the static structure of Remdesivir binding to RdRp has been solved and biochemical experiments have suggested it to be a "delayed chain terminator", the underlying molecular mechanisms is not fully understood. Here, we performed all-atom molecular dynamics (MD) simulations with an accumulated simulation time of 24 microseconds to elucidate the inhibitory mechanism of Remdesivir on nucleotide addition and proofreading. We found that when Remdesivir locates at an upstream site in RdRp, the 1'-cyano group experiences electrostatic interactions with a salt bridge (Asp865-Lys593), which subsequently halts translocation. Our findings can supplement the current understanding of the delayed chain termination exerted by Remdesivir and provide an alternative molecular explanation about Remdesivir's inhibitory mechanism. Such inhibition also reduces the likelihood of Remdesivir to be cleaved by ExoN acting on 3'-terminal nucleotides. Furthermore, our study also suggests that Remdesivir's 1'-cyano group can disrupt the cleavage site of ExoN via steric interactions, leading to a further reduction in the cleavage efficiency. Our work provides plausible and novel mechanisms at the molecular level of how Remdesivir inhibits viral RNA replication, and our findings may guide rational design for new treatments of COVID-19 targeting viral replication.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Cianetos/química , Nucleotídeos/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/fisiologia , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Monofosfato de Adenosina/farmacologia , Monofosfato de Adenosina/uso terapêutico , Alanina/química , Alanina/metabolismo , Alanina/farmacologia , Alanina/uso terapêutico , COVID-19/tratamento farmacológico , COVID-19/patologia , COVID-19/virologia , Domínio Catalítico , Humanos , Simulação de Dinâmica Molecular , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Ribose/química , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/metabolismo , Eletricidade Estática , Replicação Viral/efeitos dos fármacos
6.
Sci Rep ; 11(1): 6248, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33737523

RESUMO

The outbreak of a novel febrile respiratory disease called COVID-19, caused by a newfound coronavirus SARS-CoV-2, has brought a worldwide attention. Prioritizing approved drugs is critical for quick clinical trials against COVID-19. In this study, we first manually curated three Virus-Drug Association (VDA) datasets. By incorporating VDAs with the similarity between drugs and that between viruses, we constructed a heterogeneous Virus-Drug network. A novel Random Walk with Restart method (VDA-RWR) was then developed to identify possible VDAs related to SARS-CoV-2. We compared VDA-RWR with three state-of-the-art association prediction models based on fivefold cross-validations (CVs) on viruses, drugs and virus-drug associations on three datasets. VDA-RWR obtained the best AUCs for the three fivefold CVs, significantly outperforming other methods. We found two small molecules coming together on the three datasets, that is, remdesivir and ribavirin. These two chemical agents have higher molecular binding energies of - 7.0 kcal/mol and - 6.59 kcal/mol with the domain bound structure of the human receptor angiotensin converting enzyme 2 (ACE2) and the SARS-CoV-2 spike protein, respectively. Interestingly, for the first time, experimental results suggested that navitoclax could be potentially applied to stop SARS-CoV-2 and remains to further validation.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Enzima de Conversão de Angiotensina 2/química , Antivirais/química , Ribavirina/química , Glicoproteína da Espícula de Coronavírus/química , Monofosfato de Adenosina/química , Alanina/química , Compostos de Anilina/química , Avaliação Pré-Clínica de Medicamentos , Genoma Viral , Simulação de Acoplamento Molecular , SARS-CoV-2/genética , Sulfonamidas/química
7.
Food Chem ; 354: 129360, 2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-33735697

RESUMO

In this study, effects of low-intensity electrical currents (200, 800 and 1400 mA), ultrasound frequencies (24 and 40 kHz) and their combinations were applied at the duration period of 2, 4, 6, 8, and 10 min for the degradation of captan, thiamethoxam and metalaxyl residues in lettuce samples. Residues of the pesticides were determined by gas chromatography with tandem mass spectrometry and electron capture detector. The results indicated that the combination of low-intensity electrical current and ultrasound was found to be effective for the reduction of the pesticides. The most effective combination was obtained to be current of 1400 mA and ultrasound frequency of 24 kHz at 10 min. Under this circumstance, 92.57, 81.99 and 93.09% of captan, thiamethoxam and metalaxyl residues were decreased, respectively. The findings suggest that the combination of low-intensity electrical current and ultrasound applications has an important potential for the degradation of pesticide residues.


Assuntos
Contaminação de Alimentos/análise , Alface/química , Resíduos de Praguicidas/análise , Alanina/análogos & derivados , Alanina/análise , Alanina/química , Captana/análise , Captana/química , Eletricidade , Cromatografia Gasosa-Espectrometria de Massas/métodos , Alface/metabolismo , Resíduos de Praguicidas/química , Sonicação , Tiametoxam/análise , Tiametoxam/química
8.
Mol Cell ; 81(7): 1548-1552.e4, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33631104

RESUMO

Remdesivir is a nucleoside analog approved by the US FDA for treatment of COVID-19. Here, we present a 3.9-Å-resolution cryo-EM reconstruction of a remdesivir-stalled RNA-dependent RNA polymerase complex, revealing full incorporation of 3 copies of remdesivir monophosphate (RMP) and a partially incorporated fourth RMP in the active site. The structure reveals that RMP blocks RNA translocation after incorporation of 3 bases following RMP, resulting in delayed chain termination, which can guide the rational design of improved antiviral drugs.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/química , RNA Viral/química , RNA Polimerase Dependente de RNA/química , SARS-CoV-2/fisiologia , Replicação Viral , Monofosfato de Adenosina/química , Monofosfato de Adenosina/uso terapêutico , Alanina/química , Alanina/uso terapêutico , Antivirais/uso terapêutico , Domínio Catalítico , Humanos , Proteínas Virais
9.
Biochem Biophys Res Commun ; 538: 145-150, 2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33388129

RESUMO

Human coronaviruses (HCoV) were discovered in the 1960s and were originally thought to cause only mild upper respiratory tract diseases in immunocompetent hosts. This view changed since the beginning of this century, with the 2002 SARS (severe acute respiratory syndrome) epidemic and the 2012 MERS (Middle East respiratory syndrome) outbreak, two zoonotic infections that resulted in mortality rates of approximately 10% and 35%, respectively. Despite the importance of these pathogens, no approved antiviral drugs for the treatment of human coronavirus infections became available. However, remdesivir, a nucleotide analogue prodrug originally developed for the treatment of Ebola virus, was found to inhibit the replication of a wide range of human and animal coronaviruses in vitro and in preclinical studies. It is therefore not surprising that when the highly pathogenic SARS-CoV-2 coronavirus emerged in late 2019 in China, causing global health concern due to the virus strong human-to-human transmission ability, remdesivir was one of the first clinical candidates that received attention. After in vitro studies had shown its antiviral activity against SARS-CoV-2, and a first patient was successfully treated with the drug in the USA, a number of trials on remdesivir were initiated. Several had encouraging results, particularly the ACTT-1 double blind, randomized, and placebo controlled trial that has shown shortening of the time to recovery in hospitalized patients treated with remdesivir. The results of other trials were instead negative. Here, we provide an overview of remdesivir discovery, molecular mechanism of action, and initial and current clinical studies on its efficacy.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais , COVID-19/tratamento farmacológico , Descoberta de Drogas , Doença pelo Vírus Ebola/tratamento farmacológico , Monofosfato de Adenosina/química , Monofosfato de Adenosina/isolamento & purificação , Monofosfato de Adenosina/uso terapêutico , Alanina/química , Alanina/isolamento & purificação , Alanina/uso terapêutico , Antivirais/química , Antivirais/isolamento & purificação , Antivirais/uso terapêutico , Humanos
10.
BMC Complement Med Ther ; 21(1): 41, 2021 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-33478471

RESUMO

BACKGROUND: The latest coronavirus SARS-CoV-2, discovered in China and rapidly spread Worldwide. COVID-19 affected millions of people and killed hundreds of thousands worldwide. There are many ongoing studies investigating drug(s) suitable for preventing and/or treating this pandemic; however, there are no specific drugs or vaccines available to treat or prevent SARS-CoV-2 as of today. METHODS: Fifty-eight fragrance materials, which are classified as allergen fragrance molecules, were selected and used in this study. Docking simulations were carried out using four functional proteins; the Covid19 Main Protase (MPro), Receptor binding domain (RBD) of spike protein, Nucleocapsid, and host Bromodomain protein (BRD2), as target macromolecules. Three different software, AutoDock, AutoDock Vina (Vina), and Molegro Virtual Docker (MVD), running a total of four different docking protocol with optimized energy functions were used. Results were compared with the five molecules reported in the literature as potential drugs against COVID-19. Virtual screening was carried out using Vina, molecules satisfying our cut-off (- 6.5 kcal/mol) binding affinity was confirmed by MVD. Selected molecules were analyzed using the flexible docking protocol of Vina and AutoDock default settings. RESULTS: Ten out of 58 allergen fragrance molecules were selected for further docking studies. MPro and BRD2 are potential targets for the tested allergen fragrance molecules, while RBD and Nucleocapsid showed weak binding energies. According to AutoDock results, three molecules, Benzyl Cinnamate, Dihydroambrettolide, and Galaxolide, had good binding affinities to BRD2. While Dihydroambrettolide and Galaxolide showed the potential to bind to MPro, Sclareol and Vertofix had the best calculated binding affinities to this target. When the flexible docking results analyzed, all the molecules tested had better calculated binding affinities as expected. Benzyl Benzoate and Benzyl Salicylate showed good binding affinities to BRD2. In the case of MPro, Sclareol had the lowest binding affinity among all the tested allergen fragrance molecules. CONCLUSION: Allergen fragrance molecules are readily available, cost-efficient, and shown to be safe for human use. Results showed that several of these molecules had comparable binding affinities as the potential drug molecules reported in the literature to target proteins. Thus, these allergen molecules at correct doses could have significant health benefits.


Assuntos
Alérgenos/química , Alérgenos/imunologia , COVID-19/tratamento farmacológico , COVID-19/imunologia , Odorantes , Perfumes/química , SARS-CoV-2/química , SARS-CoV-2/imunologia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Alanina/análogos & derivados , Alanina/química , Alanina/metabolismo , Alérgenos/administração & dosagem , Alérgenos/uso terapêutico , Benzopiranos/química , Benzopiranos/metabolismo , Compostos de Benzil/química , Compostos de Benzil/metabolismo , Cinamatos/química , Cinamatos/metabolismo , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/metabolismo , Proteínas do Nucleocapsídeo de Coronavírus/química , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Diterpenos/química , Diterpenos/metabolismo , Avaliação Pré-Clínica de Medicamentos , Humanos , Ligantes , Simulação de Acoplamento Molecular , Perfumes/administração & dosagem , Perfumes/uso terapêutico , Fosfoproteínas/química , Fosfoproteínas/metabolismo , SARS-CoV-2/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
11.
Nat Commun ; 12(1): 150, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33420011

RESUMO

Novel bacterial type II topoisomerase inhibitors (NBTIs) stabilize single-strand DNA cleavage breaks by DNA gyrase but their exact mechanism of action has remained hypothetical until now. We have designed a small library of NBTIs with an improved DNA gyrase-binding moiety resulting in low nanomolar inhibition and very potent antibacterial activity. They stabilize single-stranded cleavage complexes and, importantly, we have obtained the crystal structure where an NBTI binds gyrase-DNA in a single conformation lacking apparent static disorder. This directly proves the previously postulated NBTI mechanism of action and shows that they stabilize single-strand cleavage through asymmetric intercalation with a shift of the scissile phosphate. This crystal stucture shows that the chlorine forms a halogen bond with the backbone carbonyls of the two symmetry-related Ala68 residues. To the best of our knowledge, such a so-called symmetrical bifurcated halogen bond has not been identified in a biological system until now.


Assuntos
Antibacterianos/farmacologia , Cloro/metabolismo , DNA Girase/metabolismo , Inibidores da Topoisomerase II/farmacologia , Alanina/química , Alanina/metabolismo , Antibacterianos/química , Cristalografia por Raios X , DNA Girase/química , DNA Topoisomerases Tipo II , DNA de Cadeia Simples/metabolismo , Desenho de Fármacos , Canal de Potássio ERG1/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Células Hep G2 , Células Endoteliais da Veia Umbilical Humana , Humanos , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Quinolinas/química , Quinolinas/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/enzimologia , Inibidores da Topoisomerase II/química
12.
Biophys J ; 120(6): 1085-1096, 2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33460600

RESUMO

This work builds upon the record-breaking speed and generous immediate release of new experimental three-dimensional structures of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins and complexes, which are crucial to downstream vaccine and drug development. We have surveyed those structures to catch the occasional errors that could be significant for those important uses and for which we were able to provide demonstrably higher-accuracy corrections. This process relied on new validation and correction methods such as CaBLAM and ISOLDE, which are not yet in routine use. We found such important and correctable problems in seven early SARS-CoV-2 structures. Two of the structures were soon superseded by new higher-resolution data, confirming our proposed changes. For the other five, we emailed the depositors a documented and illustrated report and encouraged them to make the model corrections themselves and use the new option at the worldwide Protein Data Bank for depositors to re-version their coordinates without changing the Protein Data Bank code. This quickly and easily makes the better-accuracy coordinates available to anyone who examines or downloads their structure, even before formal publication. The changes have involved sequence misalignments, incorrect RNA conformations near a bound inhibitor, incorrect metal ligands, and cis-trans or peptide flips that prevent good contact at interaction sites. These improvements have propagated into nearly all related structures done afterward. This process constitutes a new form of highly rigorous peer review, which is actually faster and more strict than standard publication review because it has access to coordinates and maps; journal peer review would also be strengthened by such access.


Assuntos
Revisão por Pares , SARS-CoV-2/química , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/química , Alanina/farmacologia , Anticorpos Antivirais , Domínio Catalítico , RNA Polimerases Dirigidas por DNA/metabolismo , Humanos , Modelos Moleculares , Nucleocapsídeo/química , Fosfoproteínas/química , Proteínas de Ligação a RNA/química , SARS-CoV-2/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Zinco/metabolismo
13.
J Food Sci ; 86(2): 563-570, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33438215

RESUMO

Takuan-zuke is a traditional Japanese fermented pickle, prepared by dehydration of radish root (daikon) by salt-pressing or sun-drying followed by aging with salt. We previously reported that alanine, proline, and γ-aminobutyric acid (GABA) accumulate during daikon dehydration, whereas the level of glutamic acid, their precursor, decreases. We have also reported that dehydration and salt-aging markedly influence the dynamics of free amino acids. In this study, we quantitatively analyzed free amino acid levels, enzyme activity, and gene expression to characterize takuan-zuke amino acid metabolism. Enzyme activities related to alanine, proline, GABA, and glutamic acid metabolism were sustained during dehydration. Moreover, genes encoding alanine, proline, and GABA synthases (ALT1, P5CS1, and GAD4) were significantly upregulated during dehydration. These effects may represent cellular stress responses to the dehydration process. The biological response of daikon contributes to the healthy functional aspects that characterize takuan-zuke. These findings could guide the selection of suitable vegetable varieties to produce pickled vegetables with health-promoting properties. PRACTICAL APPLICATION: The fermented pickle takuan-zuke, prepared by dehydration of radish root (daikon), accumulates amino acids, such as alanine, proline, and GABA, during preparation that provide taste and health benefits. In this study, the aforementioned amino acids were found to accumulate because of the stress response of daikon during the dehydration process and not because of the action of microorganisms during fermentation. Takuan-zuke processing is a method for improving the nutrition of daikon.


Assuntos
Alanina/metabolismo , Ácido Glutâmico/metabolismo , Prolina/metabolismo , Raphanus/química , Ácido gama-Aminobutírico/metabolismo , 4-Aminobutirato Transaminase/metabolismo , Alanina/química , Dessecação , Manipulação de Alimentos , Glutamato-Amônia Ligase/metabolismo , Ácido Glutâmico/química , Concentração de Íons de Hidrogênio , Raízes de Plantas , Reação em Cadeia da Polimerase , Prolina/química , RNA de Plantas , Cloreto de Sódio/metabolismo , Ácido gama-Aminobutírico/química
14.
Artigo em Inglês | MEDLINE | ID: mdl-33486215

RESUMO

Nascent proteome presents dynamic changes in response to a certain stimulus. Thus, monitoring nascent proteome is critical to uncovering the involved biological mechanism. But the low-abundance of nascent proteome against an overwhelming pre-existing proteome limits its identification and quantification. Herein, we present a novel strategy to enrich nascent proteome from whole cell lysate for further analysis by mass spectrometry. We employed a terminal alkyne and disulfide functionalized agarose resin to capture nascent proteome which had been labeled by L-azidohomoalanine. Results from the western blot, silver staining and pulse metabolic labeling suggested that the nascent proteome could be enriched efficiently. Applied to Hela cells, the method identified about 700 nascent proteins with good correlation with previous reports. The above indicates that our strategy can be used to reveal the proteome dynamics of biological processes.


Assuntos
Alanina/análogos & derivados , Proteínas/análise , Sefarose/química , Alanina/química , Alcinos/química , Cromatografia Líquida , Química Click , Dissulfetos/química , Células HeLa , Humanos , Proteínas/química , Proteínas/metabolismo , Proteoma/análise , Proteoma/química , Proteoma/metabolismo , Espectrometria de Massas em Tandem
15.
Acta Pharm ; 71(2): 163-174, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33151166

RESUMO

The current outbreak of novel coronavirus (COVID-19) infections urges the need to identify potential therapeutic agents. Therefore, the repurposing of FDA-approved drugs against today's diseases involves the use of de-risked compounds with potentially lower costs and shorter development timelines. In this study, the recently resolved X-ray crystallographic structure of COVID-19 main protease (Mpro) was used to generate a pharmacophore model and to conduct a docking study to capture antiviral drugs as new promising COVID-19 main protease inhibitors. The developed pharmacophore successfully captured five FDA-approved antiviral drugs (lopinavir, remdesivir, ritonavir, saquinavir and raltegravir). The five drugs were successfully docked into the binding site of COVID-19 Mpro and showed several specific binding interactions that were comparable to those tying the co-crystallized inhibitor X77 inside the binding site of COVID-19 Mpro. Three of the captured drugs namely, remdesivir, lopinavir and ritonavir, were reported to have promising results in COVID-19 treatment and therefore increases the confidence in our results. Our findings suggest an additional possible mechanism of action for remdesivir as an antiviral drug inhibiting COVID-19 Mpro. Additionally, a combination of structure-based pharmacophore modeling with a docking study is expected to facilitate the discovery of novel COVID-19 Mpro inhibitors.


Assuntos
Infecções por Coronavirus/enzimologia , Pneumonia Viral/enzimologia , Inibidores de Proteases/farmacologia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Monofosfato de Adenosina/farmacologia , Monofosfato de Adenosina/uso terapêutico , Alanina/análogos & derivados , Alanina/química , Alanina/farmacologia , Alanina/uso terapêutico , Antivirais/química , Antivirais/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Cristalografia por Raios X , Descoberta de Drogas/métodos , Reposicionamento de Medicamentos , Humanos , Modelos Químicos , Simulação de Acoplamento Molecular , Estrutura Molecular , Pandemias , Pneumonia Viral/tratamento farmacológico , Inibidores de Proteases/química , Relação Estrutura-Atividade
16.
SAR QSAR Environ Res ; 31(11): 857-867, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33100032

RESUMO

A novel coronavirus recently identified in Wuhan, China (2019-nCoV) has resulted in an increasing number of patients globally, and has become a highly lethal pathogenic member of the coronavirus family affecting humans. 2019-nCoV has established itself as one of the most threatening pandemics that human beings have faced, and therefore analysis and evaluation of all possible responses against infection is required. One such strategy includes utilizing the knowledge gained from the SARS and MERS outbreaks regarding existing antivirals. Indicating a potential for success, one of the drugs, remdesivir, under repurposing studies, has shown positive results in initial clinical studies. Therefore, in the current work, the authors have attempted to utilize the remdesivir-RdRp complex - RdRp (RNA-dependent RNA polymerase) being the putative target for remdesivir - to screen a library of the already reported RdRp inhibitor database. Further clustering on the basis of structural features and scoring refinement was performed to filter out false positive hits. Finally, molecular dynamics simulation was carried out to validate the identification of hits as RdRp inhibitors against novel coronavirus 2019-nCoV. The results yielded two putative hits which can inhibit RdRp with better potency than remdesivir, subject to further biological evaluation.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/farmacologia , Simulação de Acoplamento Molecular , /antagonistas & inibidores , Monofosfato de Adenosina/química , Monofosfato de Adenosina/farmacologia , Alanina/química , Alanina/farmacologia , Antivirais/química , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/enzimologia , Infecções por Coronavirus/tratamento farmacológico , Pandemias , Pneumonia Viral , Relação Quantitativa Estrutura-Atividade , Proteínas Virais/efeitos dos fármacos
17.
Sci Rep ; 10(1): 16577, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33024223

RESUMO

SARS-CoV-2 is responsible for COVID-19, resulting in the largest pandemic in over a hundred years. After examining the molecular structures and activities of hepatitis C viral inhibitors and comparing hepatitis C virus and coronavirus replication, we previously postulated that the FDA-approved hepatitis C drug EPCLUSA (Sofosbuvir/Velpatasvir) might inhibit SARS-CoV-2. We subsequently demonstrated that Sofosbuvir triphosphate is incorporated by the relatively low fidelity SARS-CoV and SARS-CoV-2 RNA-dependent RNA polymerases (RdRps), serving as an immediate polymerase reaction terminator, but not by a host-like high fidelity DNA polymerase. Other investigators have since demonstrated the ability of Sofosbuvir to inhibit SARS-CoV-2 replication in lung and brain cells; additionally, COVID-19 clinical trials with EPCLUSA and with Sofosbuvir plus Daclatasvir have been initiated in several countries. SARS-CoV-2 has an exonuclease-based proofreader to maintain the viral genome integrity. Any effective antiviral targeting the SARS-CoV-2 RdRp must display a certain level of resistance to this proofreading activity. We report here that Sofosbuvir terminated RNA resists removal by the exonuclease to a substantially higher extent than RNA terminated by Remdesivir, another drug being used as a COVID-19 therapeutic. These results offer a molecular basis supporting the current use of Sofosbuvir in combination with other drugs in COVID-19 clinical trials.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Exonucleases/metabolismo , Pneumonia Viral/tratamento farmacológico , Pró-Fármacos/farmacologia , RNA Viral/efeitos dos fármacos , Sofosbuvir/farmacologia , Monofosfato de Adenosina/química , Monofosfato de Adenosina/farmacologia , Monofosfato de Adenosina/uso terapêutico , Alanina/química , Alanina/farmacologia , Alanina/uso terapêutico , Antivirais/química , Antivirais/uso terapêutico , Betacoronavirus/enzimologia , Infecções por Coronavirus/virologia , Descoberta de Drogas/métodos , Reposicionamento de Medicamentos/métodos , Hepacivirus/efeitos dos fármacos , Hepacivirus/enzimologia , Hepatite C/tratamento farmacológico , Hepatite C/virologia , Humanos , Pandemias , Pneumonia Viral/virologia , Pró-Fármacos/uso terapêutico , RNA Viral/química , RNA Viral/metabolismo , /metabolismo , Sofosbuvir/química , Sofosbuvir/uso terapêutico , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
18.
Org Lett ; 22(21): 8430-8435, 2020 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-33085486

RESUMO

We report an alternative approach to the unnatural nucleobase fragment seen in remdesivir (Veklury). Remdesivir displays broad-spectrum antiviral activity and is currently being evaluated in Phase III clinical trials to treat patients with COVID-19. Our route relies on the formation of a cyanoamidine intermediate, which undergoes Lewis acid-mediated cyclization to yield the desired nucleobase. The approach is strategically distinct from prior routes and could further enable the synthesis of remdesivir and other small-molecule therapeutics.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Amidinas/química , Antivirais/química , Antivirais/síntese química , Monofosfato de Adenosina/síntese química , Monofosfato de Adenosina/química , Monofosfato de Adenosina/uso terapêutico , Alanina/síntese química , Alanina/química , Alanina/uso terapêutico , Antivirais/uso terapêutico , Técnicas de Química Sintética , Infecções por Coronavirus/tratamento farmacológico , Ciclização , Pandemias , Pneumonia Viral/tratamento farmacológico
19.
Nat Commun ; 11(1): 4899, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32994420

RESUMO

Chemical synthesis of amino acids from renewable sources is an alternative route to the current processes based on fermentation. Here, we report visible-light-driven amination of biomass-derived α-hydroxyl acids and glucose into amino acids using NH3 at 50 °C. Ultrathin CdS nanosheets are identified as an efficient and stable catalyst, exhibiting an order of magnitude higher activity towards alanine production from lactic acid compared to commercial CdS as well as CdS nanoobjects bearing other morphologies. Its unique catalytic property is attributed mainly to the preferential formation of oxygen-centered radicals to promote α-hydroxyl acids conversion to α-keto acids, and partially to the poor H2 evolution which is an undesired side reaction. Encouragingly, a number of amino acids are prepared using the current protocol, and one-pot photocatalytic conversion of glucose to alanine is also achieved. This work offers an effective catalytic system for amino acid synthesis from biomass feedstocks under mild conditions.


Assuntos
Alanina/síntese química , Compostos de Cádmio/química , Sulfetos/química , Alanina/química , Biomassa , Catálise/efeitos da radiação , Hidrogênio/química , Ácido Láctico/química , Luz , Modelos Químicos , Oxigênio/química
20.
Nature ; 585(7826): 530-537, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32968259

RESUMO

Post-translational modifications (PTMs) greatly expand the structures and functions of proteins in nature1,2. Although synthetic protein functionalization strategies allow mimicry of PTMs3,4, as well as formation of unnatural protein variants with diverse potential functions, including drug carrying5, tracking, imaging6 and partner crosslinking7, the range of functional groups that can be introduced remains limited. Here we describe the visible-light-driven installation of side chains at dehydroalanine residues in proteins through the formation of carbon-centred radicals that allow C-C bond formation in water. Control of the reaction redox allows site-selective modification with good conversions and reduced protein damage. In situ generation of boronic acid catechol ester derivatives generates RH2C• radicals that form the native (ß-CH2-γ-CH2) linkage of natural residues and PTMs, whereas in situ potentiation of pyridylsulfonyl derivatives by Fe(II) generates RF2C• radicals that form equivalent ß-CH2-γ-CF2 linkages bearing difluoromethylene labels. These reactions are chemically tolerant and incorporate a wide range of functionalities (more than 50 unique residues/side chains) into diverse protein scaffolds and sites. Initiation can be applied chemoselectively in the presence of sensitive groups in the radical precursors, enabling installation of previously incompatible side chains. The resulting protein function and reactivity are used to install radical precursors for homolytic on-protein radical generation; to study enzyme function with natural, unnatural and CF2-labelled post-translationally modified protein substrates via simultaneous sensing of both chemo- and stereoselectivity; and to create generalized 'alkylator proteins' with a spectrum of heterolytic covalent-bond-forming activity (that is, reacting diversely with small molecules at one extreme or selectively with protein targets through good mimicry at the other). Post-translational access to such reactions and chemical groups on proteins could be useful in both revealing and creating protein function.


Assuntos
Luz , Processamento de Proteína Pós-Traducional/efeitos da radiação , Proteínas/química , Proteínas/metabolismo , Alanina/análogos & derivados , Alanina/química , Alanina/metabolismo , Sítios de Ligação , Carbono/química , Carbono/metabolismo , Enzimas/química , Enzimas/metabolismo , Ésteres/síntese química , Ésteres/química , Células HeLa , Humanos , Hidrocarbonetos Fluorados/química , Hidrocarbonetos Fluorados/metabolismo , Indicadores e Reagentes/química , Oxirredução , Processos Fotoquímicos/efeitos da radiação , Domínios e Motivos de Interação entre Proteínas
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