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1.
J Hazard Mater ; 416: 125877, 2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-34492817

RESUMO

Ag/NaBiO3 with dual active sites and high capacitance was prepared by the photo-deposition method. Upon light illumination, the reduction of Ag+ to Ag, the introduction of oxygen vacancies, and the electron storage in Ag nanoparticles simultaneously happened. NO, and O2 adsorbed and activated at Ag site and oxygen vacancy site, respectively, to produce active ON* and •O2- radical species. The increased concentrations of the active oxygen species and the pre-oxidation of NO resulted in the enhanced NO removal with inhibited production of NO2. Moreover, the high capacitance of Ag and the continuous charge transfer from defective NaBiO3 to Ag offered the enhanced and long-lasting dark catalytic activity of the Ag/NaBiO3. The stored electrons in Ag were directly released in dark to decompose methyl orange and/or tetracycline. This work provides a novel idea of designing and preparing a multifunctional catalytic material for environmental cleaning.


Assuntos
Poluentes Ambientais , Nanopartículas Metálicas , Domínio Catalítico , Oxirredução , Prata
2.
Molecules ; 26(17)2021 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-34500643

RESUMO

Mammalian cell surfaces are modified with complex arrays of glycans that play major roles in health and disease. Abnormal glycosylation is a hallmark of cancer; terminal sialic acid and fucose in particular have high levels in tumor cells, with positive implications for malignancy. Increased sialylation and fucosylation are due to the upregulation of a set of sialyltransferases (STs) and fucosyltransferases (FUTs), which are potential drug targets in cancer. In the past, several advances in glycostructural biology have been made with the determination of crystal structures of several important STs and FUTs in mammals. Additionally, how the independent evolution of STs and FUTs occurred with a limited set of global folds and the diverse modular ability of catalytic domains toward substrates has been elucidated. This review highlights advances in the understanding of the structural architecture, substrate binding interactions, and catalysis of STs and FUTs in mammals. While this general understanding is emerging, use of this information to design inhibitors of STs and FUTs will be helpful in providing further insights into their role in the manifestation of cancer and developing targeted therapeutics in cancer.


Assuntos
Fucosiltransferases/química , Fucosiltransferases/metabolismo , Mamíferos/metabolismo , Sialiltransferases/química , Sialiltransferases/metabolismo , Animais , Catálise , Domínio Catalítico/fisiologia , Glicosilação , Humanos
4.
Int J Mol Sci ; 22(17)2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-34502335

RESUMO

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been identified as the pathogen responsible for the outbreak of a severe, rapidly developing pneumonia (Coronavirus disease 2019, COVID-19). The virus enzyme, called 3CLpro or main protease (Mpro), is essential for viral replication, making it a most promising target for antiviral drug development. Recently, we adopted the drug repurposing as appropriate strategy to give fast response to global COVID-19 epidemic, by demonstrating that the zonulin octapeptide inhibitor AT1001 (Larazotide acetate) binds Mpro catalytic domain. Thus, in the present study we tried to investigate the antiviral activity of AT1001, along with five derivatives, by cell-based assays. Our results provide with the identification of AT1001 peptide molecular framework for lead optimization step to develop new generations of antiviral agents of SARS-CoV-2 with an improved biological activity, expanding the chance for success in clinical trials.


Assuntos
Antivirais/farmacologia , Simulação de Acoplamento Molecular , Oligopeptídeos/química , Peptídeos/metabolismo , SARS-CoV-2/efeitos dos fármacos , Antivirais/química , Antivirais/metabolismo , Antivirais/uso terapêutico , Sítios de Ligação , COVID-19/tratamento farmacológico , COVID-19/virologia , Domínio Catalítico , Linhagem Celular , Citomegalovirus/efeitos dos fármacos , Reposicionamento de Medicamentos , Herpesvirus Humano 3/efeitos dos fármacos , Humanos , Simulação de Dinâmica Molecular , Peptídeos/síntese química , Peptídeos/farmacologia , Peptídeos/uso terapêutico , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/metabolismo , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/metabolismo
5.
Int J Mol Sci ; 22(17)2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-34502340

RESUMO

The SARS-CoV-2 main protease (Mpro) is one of the molecular targets for drug design. Effective vaccines have been identified as a long-term solution but the rate at which they are being administered is slow in several countries, and mutations of SARS-CoV-2 could render them less effective. Moreover, remdesivir seems to work only with some types of COVID-19 patients. Hence, the continuous investigation of new treatments for this disease is pivotal. This study investigated the inhibitory role of natural products against SARS-CoV-2 Mpro as repurposable agents in the treatment of coronavirus disease 2019 (COVID-19). Through in silico approach, selected flavonoids were docked into the active site of Mpro. The free energies of the ligands complexed with Mpro were computationally estimated using the molecular mechanics-generalized Born surface area (MM/GBSA) method. In addition, the inhibition process of SARS-CoV-2 Mpro with these ligands was simulated at 100 ns in order to uncover the dynamic behavior and complex stability. The docking results showed that the selected flavonoids exhibited good poses in the binding domain of Mpro. The amino acid residues involved in the binding of the selected ligands correlated well with the residues involved with the mechanism-based inhibitor (N3) and the docking score of Quercetin-3-O-Neohesperidoside (-16.8 Kcal/mol) ranked efficiently with this inhibitor (-16.5 Kcal/mol). In addition, single-structure MM/GBSA rescoring method showed that Quercetin-3-O-Neohesperidoside (-87.60 Kcal/mol) is more energetically favored than N3 (-80.88 Kcal/mol) and other ligands (Myricetin 3-Rutinoside (-87.50 Kcal/mol), Quercetin 3-Rhamnoside (-80.17 Kcal/mol), Rutin (-58.98 Kcal/mol), and Myricitrin (-49.22 Kcal/mol). The molecular dynamics simulation (MDs) pinpointed the stability of these complexes over the course of 100 ns with reduced RMSD and RMSF. Based on the docking results and energy calculation, together with the RMSD of 1.98 ± 0.19 Å and RMSF of 1.00 ± 0.51 Å, Quercetin-3-O-Neohesperidoside is a better inhibitor of Mpro compared to N3 and other selected ligands and can be repurposed as a drug candidate for the treatment of COVID-19. In addition, this study demonstrated that in silico docking, free energy calculations, and MDs, respectively, are applicable to estimating the interaction, energetics, and dynamic behavior of molecular targets by natural products and can be used to direct the development of novel target function modulators.


Assuntos
Produtos Biológicos/metabolismo , SARS-CoV-2/enzimologia , Proteínas da Matriz Viral/metabolismo , Sítios de Ligação , Produtos Biológicos/química , Produtos Biológicos/uso terapêutico , COVID-19/tratamento farmacológico , COVID-19/patologia , COVID-19/virologia , Domínio Catalítico , Desenho de Fármacos , Humanos , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Inibidores de Proteases/uso terapêutico , Quercetina/análogos & derivados , Quercetina/química , Quercetina/metabolismo , Quercetina/uso terapêutico , SARS-CoV-2/isolamento & purificação , Proteínas da Matriz Viral/química
6.
Nat Commun ; 12(1): 5254, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489436

RESUMO

Pdr5, a member of the extensive ABC transporter superfamily, is representative of a clinically relevant subgroup involved in pleiotropic drug resistance. Pdr5 and its homologues drive drug efflux through uncoupled hydrolysis of nucleotides, enabling organisms such as baker's yeast and pathogenic fungi to survive in the presence of chemically diverse antifungal agents. Here, we present the molecular structure of Pdr5 solved with single particle cryo-EM, revealing details of an ATP-driven conformational cycle, which mechanically drives drug translocation through an amphipathic channel, and a clamping switch within a conserved linker loop that acts as a nucleotide sensor. One half of the transporter remains nearly invariant throughout the cycle, while its partner undergoes changes that are transmitted across inter-domain interfaces to support a peristaltic motion of the pumped molecule. The efflux model proposed here rationalises the pleiotropic impact of Pdr5 and opens new avenues for the development of effective antifungal compounds.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Trifosfato de Adenosina/metabolismo , Domínio Catalítico , Microscopia Crioeletrônica , Detergentes/química , Farmacorresistência Fúngica/genética , Pleiotropia Genética , Hidrólise , Mutação , Conformação Proteica , Domínios Proteicos , Rodaminas/química , Rodaminas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Vanadatos/química , Vanadatos/metabolismo
7.
Nat Commun ; 12(1): 5277, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489474

RESUMO

The pyruvate dehydrogenase complex (PDHc) links glycolysis to the citric acid cycle by converting pyruvate into acetyl-coenzyme A. PDHc encompasses three enzymatically active subunits, namely pyruvate dehydrogenase, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase. Dihydrolipoyl transacetylase is a multidomain protein comprising a varying number of lipoyl domains, a peripheral subunit-binding domain, and a catalytic domain. It forms the structural core of the complex, provides binding sites for the other enzymes, and shuffles reaction intermediates between the active sites through covalently bound lipoyl domains. The molecular mechanism by which this shuttling occurs has remained elusive. Here, we report a cryo-EM reconstruction of the native E. coli dihydrolipoyl transacetylase core in a resting state. This structure provides molecular details of the assembly of the core and reveals how the lipoyl domains interact with the core at the active site.


Assuntos
Proteínas de Escherichia coli/química , Complexo Piruvato Desidrogenase/química , Complexo Piruvato Desidrogenase/metabolismo , Domínio Catalítico , Microscopia Crioeletrônica , Di-Hidrolipoil-Lisina-Resíduo Acetiltransferase/química , Di-Hidrolipoil-Lisina-Resíduo Acetiltransferase/metabolismo , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Lisina/análogos & derivados , Lisina/química , Lisina/metabolismo , Modelos Moleculares , Domínios Proteicos , Complexo Piruvato Desidrogenase/isolamento & purificação , Ácido Tióctico/análogos & derivados , Ácido Tióctico/química , Ácido Tióctico/metabolismo
8.
Chem Commun (Camb) ; 57(72): 9096-9099, 2021 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-34498651

RESUMO

We present a detailed computational analysis of the binding mode and reactivity of the novel oral inhibitor PF-07321332 developed against the SARS-CoV-2 3CL protease. Alchemical free energy calculations suggest that positions P3 and P4 could be susceptible to improvement in order to get a larger binding strength. QM/MM simulations unveil the reaction mechanism for covalent inhibition, showing that the nitrile warhead facilitates the recruitment of a water molecule for the proton transfer step.


Assuntos
Proteases 3C de Coronavírus/antagonistas & inibidores , Simulação de Dinâmica Molecular , Nitrilas/química , Inibidores de Proteases/química , SARS-CoV-2/enzimologia , Sítios de Ligação , COVID-19/patologia , COVID-19/virologia , Domínio Catalítico , Proteases 3C de Coronavírus/metabolismo , Humanos , Lactamas/química , Lactamas/metabolismo , Leucina/química , Leucina/metabolismo , Nitrilas/metabolismo , Prolina/química , Prolina/metabolismo , Inibidores de Proteases/metabolismo , Teoria Quântica , SARS-CoV-2/isolamento & purificação , Termodinâmica
9.
Molecules ; 26(17)2021 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-34500777

RESUMO

Human neutrophil elastase (HNE) is a uniquely destructive serine protease with the ability to unleash a wave of proteolytic activity by destroying the inhibitors of other proteases. Although this phenomenon forms an important part of the innate immune response to invading pathogens, it is responsible for the collateral host tissue damage observed in chronic conditions such as chronic obstructive pulmonary disease (COPD), and in more acute disorders such as the lung injuries associated with COVID-19 infection. Previously, a combinatorially selected activity-based probe revealed an unexpected substrate preference for oxidised methionine, which suggests a link to oxidative pathogen clearance by neutrophils. Here we use oxidised model substrates and inhibitors to confirm this observation and to show that neutrophil elastase is specifically selective for the di-oxygenated methionine sulfone rather than the mono-oxygenated methionine sulfoxide. We also posit a critical role for ordered solvent in the mechanism of HNE discrimination between the two oxidised forms methionine residue. Preference for the sulfone form of oxidised methionine is especially significant. While both host and pathogens have the ability to reduce methionine sulfoxide back to methionine, a biological pathway to reduce methionine sulfone is not known. Taken together, these data suggest that the oxidative activity of neutrophils may create rapidly cleaved elastase "super substrates" that directly damage tissue, while initiating a cycle of neutrophil oxidation that increases elastase tissue damage and further neutrophil recruitment.


Assuntos
Imunidade Inata , Elastase de Leucócito/metabolismo , Metionina/análogos & derivados , Neutrófilos/imunologia , Biocatálise , COVID-19/imunologia , COVID-19/patologia , COVID-19/virologia , Domínio Catalítico/genética , Ensaios Enzimáticos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Elastase de Leucócito/antagonistas & inibidores , Elastase de Leucócito/genética , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Metionina/metabolismo , Simulação de Dinâmica Molecular , Infiltração de Neutrófilos , Neutrófilos/enzimologia , Oxirredução/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/patologia , SARS-CoV-2/imunologia , Especificidade por Substrato/imunologia
10.
Int J Mol Sci ; 22(17)2021 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-34502033

RESUMO

The novel coronavirus disease, caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), rapidly spreading around the world, poses a major threat to the global public health. Herein, we demonstrated the binding mechanism of PF-07321332, α-ketoamide, lopinavir, and ritonavir to the coronavirus 3-chymotrypsin-like-protease (3CLpro) by means of docking and molecular dynamic (MD) simulations. The analysis of MD trajectories of 3CLpro with PF-07321332, α-ketoamide, lopinavir, and ritonavir revealed that 3CLpro-PF-07321332 and 3CLpro-α-ketoamide complexes remained stable compared with 3CLpro-ritonavir and 3CLpro-lopinavir. Investigating the dynamic behavior of ligand-protein interaction, ligands PF-07321332 and α-ketoamide showed stronger bonding via making interactions with catalytic dyad residues His41-Cys145 of 3CLpro. Lopinavir and ritonavir were unable to disrupt the catalytic dyad, as illustrated by increased bond length during the MD simulation. To decipher the ligand binding mode and affinity, ligand interactions with SARS-CoV-2 proteases and binding energy were calculated. The binding energy of the bespoke antiviral PF-07321332 clinical candidate was two times higher than that of α-ketoamide and three times than that of lopinavir and ritonavir. Our study elucidated in detail the binding mechanism of the potent PF-07321332 to 3CLpro along with the low potency of lopinavir and ritonavir due to weak binding affinity demonstrated by the binding energy data. This study will be helpful for the development and optimization of more specific compounds to combat coronavirus disease.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Protease de Coronavírus/farmacologia , Lactamas/farmacologia , Leucina/farmacologia , Nitrilas/farmacologia , Prolina/farmacologia , Antivirais/uso terapêutico , Domínio Catalítico/efeitos dos fármacos , Proteases 3C de Coronavírus/metabolismo , Inibidores de Protease de Coronavírus/uso terapêutico , Humanos , Lactamas/uso terapêutico , Leucina/uso terapêutico , Lopinavir/farmacologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Nitrilas/uso terapêutico , Prolina/uso terapêutico , Ritonavir/farmacologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia
11.
Anal Chim Acta ; 1179: 338812, 2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34535246

RESUMO

Metal active species combined with N-doped porous carbon nanosheets usually own excellent electrochemical activity and sensing performance owing to its unique microstructure and composition. In this work, monodispersed Ni active sites anchored on N-doped porous carbon nanosheets (Ni@N-PCN) were facilely prepared via rational metal-organic frameworks (MOFs) route. Firstly, zeolitic imidazolate frameworks-8 (ZIF-8) was in situ grown on physically-exfoliated graphene nanosheets (GN) with homogeneous sandwich-like structure (ZIF-8@GN). Secondly, nickel bonded ZIF-8@GN hybrids (Ni/ZIF-8@GN) were obtained by ionic exchange reaction, and then transformed into Ni@N-PCN by high-temperature pyrolysis. Benefiting from the monodispersed Ni active sites and highly reactive N-doped porous carbon nanosheets (N-PCN), the as-prepared Ni@N-PCN hybrids displayed superior catalytic performance toward hydrogen peroxide (H2O2) sensing. As a result, a highly sensitive electrochemical sensing platform for H2O2 was fabricated with low detection limit (0.032 µM), wide detection linearity (0.2-2332.8 µM), and high sensitivity (6085 µA cm-2 mM-1). Besides, the as-developed electrochemical sensing platform was successfully applied to detect H2O2 contents in biological medicine and food specimens with satisfied results. This study will provide effective guidance for the preparation of novel metal/N-doped carbon nanomaterials and establishment of high-performance electrochemical sensors.


Assuntos
Carbono , Estruturas Metalorgânicas , Domínio Catalítico , Peróxido de Hidrogênio , Porosidade
12.
Appl Microbiol Biotechnol ; 105(16-17): 6159-6172, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34350478

RESUMO

Oleate hydratase catalyses the addition of water to the CC double bond of oleic acid to produce (R)-10-hydroxystearic acid. The enzyme requires an FAD cofactor that functions to optimise the active site structure. A wide range of unsaturated fatty acids can be hydrated at the C10 and in some cases the C13 position. The substrate scope can be expanded using 'decoy' small carboxylic acids to convert small chain alkenes to secondary alcohols, albeit at low conversion rates. Systematic protein engineering and directed evolution to widen the substrate scope and increase the conversion rate is possible, supported by new high throughput screening assays that have been developed. Multi-enzyme cascades allow the formation of a wide range of products including keto-fatty acids, secondary alcohols, secondary amines and α,ω-dicarboxylic acids. KEY POINTS: • Phylogenetically distinct oleate hydratases may exhibit mechanistic differences. • Protein engineering to improve productivity and substrate scope is possible. • Multi-enzymatic cascades greatly widen the product portfolio.


Assuntos
Hidroliases , Ácido Oleico , Catálise , Domínio Catalítico , Ácidos Graxos Insaturados , Hidroliases/genética , Hidroliases/metabolismo
13.
Science ; 373(6553)2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34437092

RESUMO

Systematic and extensive investigation of enzymes is needed to understand their extraordinary efficiency and meet current challenges in medicine and engineering. We present HT-MEK (High-Throughput Microfluidic Enzyme Kinetics), a microfluidic platform for high-throughput expression, purification, and characterization of more than 1500 enzyme variants per experiment. For 1036 mutants of the alkaline phosphatase PafA (phosphate-irrepressible alkaline phosphatase of Flavobacterium), we performed more than 670,000 reactions and determined more than 5000 kinetic and physical constants for multiple substrates and inhibitors. We uncovered extensive kinetic partitioning to a misfolded state and isolated catalytic effects, revealing spatially contiguous regions of residues linked to particular aspects of function. Regions included active-site proximal residues but extended to the enzyme surface, providing a map of underlying architecture not possible to derive from existing approaches. HT-MEK has applications that range from understanding molecular mechanisms to medicine, engineering, and design.


Assuntos
Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Fosfatase Alcalina/antagonistas & inibidores , Fosfatase Alcalina/química , Biocatálise , Domínio Catalítico , Flavobacterium/enzimologia , Hidrólise , Cinética , Microfluídica , Modelos Moleculares , Mutação , Oxigênio/metabolismo , Fosfatos/metabolismo , Conformação Proteica , Dobramento de Proteína , Termodinâmica
14.
J Enzyme Inhib Med Chem ; 36(1): 1938-1951, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34459690

RESUMO

In this paper, bis (indol-3-yl) methanes (BIMs) were synthesised and evaluated for their inhibitory activity against α-glucosidase and α-amylase. All synthesised compounds showed potential α-glucosidase and α-amylase inhibitory activities. Compounds 5 g (IC50: 7.54 ± 1.10 µM), 5e (IC50: 9.00 ± 0.97 µM), and 5 h (IC50: 9.57 ± 0.62 µM) presented strongest inhibitory activities against α-glucosidase, that were ∼ 30 times stronger than acarbose. Compounds 5 g (IC50: 32.18 ± 1.66 µM), 5 h (IC50: 31.47 ± 1.42 µM), and 5 s (IC50: 30.91 ± 0.86 µM) showed strongest inhibitory activities towards α-amylase, ∼ 2.5 times stronger than acarbose. The mechanisms and docking simulation of the compounds were also studied. Compounds 5 g and 5 h exhibited bifunctional inhibitory activity against these two enzymes. Furthermore, compounds showed no toxicity against 3T3-L1 cells and HepG2 cells.HighlightsA series of bis (indol-3-yl) methanes (BIMs) were synthesised and evaluated inhibitory activities against α-glucosidase and α-amylase.Compound 5g exhibited promising activity (IC50 = 7.54 ± 1.10 µM) against α-glucosidase.Compound 5s exhibited promising activity (IC50 = 30.91 ± 0.86 µM) against α-amylase.In silico studies were performed to confirm the binding interactions of synthetic compounds with the enzyme active site.


Assuntos
Inibidores de Glicosídeo Hidrolases/síntese química , Indóis/síntese química , Metano/síntese química , alfa-Amilases/metabolismo , alfa-Glucosidases/metabolismo , Células 3T3 , Acarbose/química , Animais , Domínio Catalítico , Inibidores de Glicosídeo Hidrolases/metabolismo , Células Hep G2 , Humanos , Cinética , Metano/metabolismo , Camundongos , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade
15.
J Phys Chem Lett ; 12(32): 7777-7782, 2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34374547

RESUMO

Enzyme catalysis achieves tremendous rate accelerations. Enzyme reaction centers provide a constraint geometry that preferentially binds an activated form of the substrate and thus lowers the energy barrier. However, this transition state picture neglects the flexibility of proteins and its role in enzymatic catalysis. Especially for proton transfer reactions, it has been suggested that motions of the protein modulate the donor-acceptor distance and prepare a tunneling-ready state. We report the detection of frequency fluctuations of an azide anion (N3-) bound in the active site of the protein carbonic anhydrase II, where a low-frequency mode of the protein has been proposed to facilitate proton transfer over two water molecules during the catalyzed reaction. 2D-IR spectroscopy resolves an underdamped low-frequency mode at about 1 THz (30 cm-1). We find its frequency to be viscosity- and temperature-dependent and to decrease by 6 cm-1 between 230 and 320 K, reporting the softening of the mode's potential.


Assuntos
Anidrase Carbônica II/química , Animais , Azidas/química , Domínio Catalítico , Bovinos , Prótons , Espectrofotometria Infravermelho/métodos , Temperatura , Vibração , Viscosidade , Água/química
16.
Molecules ; 26(16)2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34443335

RESUMO

The specificity of inhibition by 6,6'-dihydroxythiobinupharidine (DTBN) on cysteine proteases was demonstrated in this work. There were differences in the extent of inhibition, reflecting active site structural-steric and biochemical differences. Cathepsin S (IC50 = 3.2 µM) was most sensitive to inhibition by DTBN compared to Cathepsin B, L and papain (IC50 = 1359.4, 13.2 and 70.4 µM respectively). DTBN is inactive for the inhibition of Mpro of SARS-CoV-2. Docking simulations suggested a mechanism of interaction that was further supported by the biochemical results. In the docking results, it was shown that the cysteine sulphur of Cathepsin S, L and B was in close proximity to the DTBN thiaspirane ring, potentially forming the necessary conditions for a nucleophilic attack to form a disulfide bond. Covalent docking and molecular dynamic simulations were performed to validate disulfide bond formation and to determine the stability of Cathepsins-DTBN complexes, respectively. The lack of reactivity of DTBN against SARS-CoV-2 Mpro was attributed to a mismatch of the binding conformation of DTBN to the catalytic binding site of Mpro. Thus, gradations in reactivity among the tested Cathepsins may be conducive for a mechanism-based search for derivatives of nupharidine against COVID-19. This could be an alternative strategy to the large-scale screening of electrophilic inhibitors.


Assuntos
Alcaloides/farmacologia , Cisteína Proteases/metabolismo , Alcaloides/química , Animais , Antivirais/farmacologia , Sítios de Ligação , COVID-19/tratamento farmacológico , COVID-19/metabolismo , Domínio Catalítico , Catepsinas/farmacologia , Linhagem Celular Tumoral , Cisteína Proteases/química , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/farmacologia , Humanos , Camundongos , Simulação de Acoplamento Molecular/métodos , Nuphar/química , Papaína/farmacologia , Extratos Vegetais/farmacologia , Ligação Proteica , SARS-CoV-2/efeitos dos fármacos
17.
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
18.
Nucleic Acids Res ; 49(15): 8796-8810, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34379778

RESUMO

During RNA elongation, the influenza A viral (IAV) RNA-dependent RNA polymerase (RdRp) residues in the active site interact with the triphosphate moiety of nucleoside triphosphate (NTP) for catalysis. The molecular mechanisms by which they control the rate and fidelity of NTP incorporation remain elusive. Here, we demonstrated through enzymology, virology and computational approaches that the R239 and K235 in the PB1 subunit of RdRp are critical to controlling the activity and fidelity of transcription. Contrary to common beliefs that high-fidelity RdRp variants exert a slower incorporation rate, we discovered a first-of-its-kind, single lysine-to-arginine mutation on K235 exhibited enhanced fidelity and activity compared with wild-type. In particular, we employed a single-turnover NTP incorporation assay for the first time on IAV RdRp to show that K235R mutant RdRp possessed a 1.9-fold increase in the transcription activity of the cognate NTP and a 4.6-fold increase in fidelity compared to wild-type. Our all-atom molecular dynamics simulations further elucidated that the higher activity is attributed to the shorter distance between K235R and the triphosphate moiety of NTP compared with wild-type. These results provide novel insights into NTP incorporation and fidelity control mechanisms, which lay the foundation for the rational design of IAV vaccine and antiviral targets.


Assuntos
Vírus da Influenza A/enzimologia , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Transcrição Genética , Proteínas Virais/química , Proteínas Virais/metabolismo , Substituição de Aminoácidos , Animais , Domínio Catalítico , Cães , Sequenciamento de Nucleotídeos em Larga Escala , Células Madin Darby de Rim Canino , Mutação , RNA Polimerase Dependente de RNA/genética , Alinhamento de Sequência , Proteínas Virais/genética
19.
J Phys Chem Lett ; 12(34): 8384-8396, 2021 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-34435784

RESUMO

This study utilizes the FMN-dependent NADH:quinone oxidoreductase from Pseudomonas aeruginosa PAO1 to investigate the effect of introducing an active site negative charge on the flavin absorption spectrum both in the absence and presence of a long-range electrostatic potential coming from solution ions. There were no observed changes in the flavin UV-visible spectrum when an active site tyrosine (Y277) becomes deprotonated in vitro. These results could only be reproduced computationally using average solvent electrostatic configuration (ASEC) QM/MM simulations that include both positive and negative solution ions. The same calculations performed with minimal ions to neutralize the total protein charge predicted that deprotonating Y277 would significantly alter the flavin absorption spectrum. Analyzing the distribution of solution ions indicated that the ions reorganize around the protein surface upon Y277 deprotonation to cancel the effect of the tyrosinate on the flavin absorption spectrum. Additional biochemical experiments were performed to test this hypothesis.


Assuntos
Absorção Fisico-Química , Flavoproteínas/química , Domínio Catalítico , Modelos Moleculares , Soluções
20.
Nat Commun ; 12(1): 5055, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417448

RESUMO

Reactive oxygen species (ROS) oxidize cellular nucleotide pools and cause double strand breaks (DSBs). Non-homologous end-joining (NHEJ) attaches broken chromosomal ends together in mammalian cells. Ribonucleotide insertion by DNA polymerase (pol) µ prepares breaks for end-joining and this is required for successful NHEJ in vivo. We previously showed that pol µ lacks discrimination against oxidized dGTP (8-oxo-dGTP), that can lead to mutagenesis, cancer, aging and human disease. Here we reveal the structural basis for proficient oxidized ribonucleotide (8-oxo-rGTP) incorporation during DSB repair by pol µ. Time-lapse crystallography snapshots of structural intermediates during nucleotide insertion along with computational simulations reveal substrate, metal and side chain dynamics, that allow oxidized ribonucleotides to escape polymerase discrimination checkpoints. Abundant nucleotide pools, combined with inefficient sanitization and repair, implicate pol µ mediated oxidized ribonucleotide insertion as an emerging source of widespread persistent mutagenesis and genomic instability.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Ribonucleotídeos/metabolismo , Adenina/metabolismo , Cálcio/metabolismo , Domínio Catalítico , Citosina/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Nucleotídeos de Desoxiguanina/química , Nucleotídeos de Desoxiguanina/metabolismo , Humanos , Cinética , Manganês/metabolismo , Modelos Moleculares , Oxirredução
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