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1.
Molecules ; 26(13)2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34208805

RESUMO

This article presents experimental evidence and computed molecular models of a potential interaction between receptor domain D5 of TrkB with the carboxyl-terminal domain of tetanus neurotoxin (Hc-TeNT). Computational simulations of a novel small cyclic oligopeptide are designed, synthesized, and tested for possible tetanus neurotoxin-D5 interaction. A hot spot of this protein-protein interaction is identified in analogy to the hitherto known crystal structures of the complex between neurotrophin and D5. Hc-TeNT activates the neurotrophin receptors, as well as its downstream signaling pathways, inducing neuroprotection in different stress cellular models. Based on these premises, we propose the Trk receptor family as potential proteic affinity receptors for TeNT. In vitro, Hc-TeNT binds to a synthetic TrkB-derived peptide and acts similar to an agonist ligand for TrkB, resulting in phosphorylation of the receptor. These properties are weakened by the mutagenesis of three residues of the predicted interaction region in Hc-TeNT. It also competes with Brain-derived neurotrophic factor, a native binder to human TrkB, for the binding to neural membranes, and for uptake in TrkB-positive vesicles. In addition, both molecules are located together In Vivo at neuromuscular junctions and in motor neurons.


Assuntos
Glicoproteínas de Membrana/química , Metaloendopeptidases/química , Fármacos Neuroprotetores/química , Oligopeptídeos/química , Receptor trkB/química , Toxina Tetânica/química , Animais , Cristalografia por Raios X , Humanos , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/farmacologia , Metaloendopeptidases/metabolismo , Metaloendopeptidases/farmacologia , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacologia , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Domínios Proteicos , Ratos , Ratos Sprague-Dawley , Receptor trkB/metabolismo , Receptor trkB/farmacologia , Toxina Tetânica/metabolismo , Toxina Tetânica/farmacologia
2.
Molecules ; 26(11)2021 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-34198944

RESUMO

Single crystal of furazolidone (FZL) has been successfully obtained, and its crystal structure has been determined. Common and distinctive features of furazolidone and nitrofurantoin (NFT) crystal packing have been discussed. Combined use of QTAIMC and Hirshfeld surface analysis allowed characterizing the non-covalent interactions in both crystals. Thermophysical characteristics and decomposition of NFT and FZL have been studied by differential scanning calorimetry (DSC), thermogravimetric analysis (TG) and mass-spectrometry. The saturated vapor pressures of the compounds have been measured using the transpiration method, and the standard thermodynamic functions of sublimation were calculated. It was revealed that the sublimation enthalpy and Gibbs energy of NFT are both higher than those for FZL, but a gain in the crystal lattice energy of NFT is leveled by an entropy increase. The solubility processes of the studied compounds in buffer solutions with pH 2.0, 7.4 and in 1-octanol was investigated at four temperatures from 298.15 to 313.15 K by the saturation shake-flask method. The thermodynamic functions of the dissolution and solvation processes of the studied compounds have been calculated based on the experimental data. Due to the fact that NFT is unstable in buffer solutions and undergoes a solution-mediated transformation from an anhydrate form to monohydrate in the solid state, the thermophysical characteristics and dissolution thermodynamics of the monohydrate were also investigated. It was demonstrated that a combination of experimental and theoretical methods allows performing an in-depth study of the relationships between the molecular and crystal structure and pharmaceutically relevant properties of nitrofuran antibiotics.


Assuntos
Antibacterianos/química , Furazolidona/química , Nitrofurantoína/química , Antibacterianos/farmacocinética , Varredura Diferencial de Calorimetria , Cristalografia por Raios X , Teoria da Densidade Funcional , Furazolidona/farmacocinética , Espectrometria de Massas , Estrutura Molecular , Nitrofurantoína/farmacocinética , Solubilidade , Termodinâmica , Termogravimetria
3.
Int J Mol Sci ; 22(13)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206574

RESUMO

Sorafenib (Sor) is an oral multi-kinase inhibitor, but its water solubility is very low. To improve its solubility, sorafenib hydrochloride hydrate, sorafenib hydrobromide and sorafenib hydrobromide hydrate were prepared in the mixed solvent of the corresponding acid solution, and tetrahydrofuran (THF). The crystal structures of sorafenib hydrochloride trihydrate (Sor·HCl.3H2O), 4-(4-{3-[4-chloro-3-(trifluoro-methyl)phenyl]ureido}phenoxy)-2-(N-methylcarbamoyl) pyridinium hydrochloride trihydrate, C21H17ClF3N4O3+·Cl-.3H2O (I), sorafenib hydrochloride monohydrate (Sor·HCl.H2O), C21H17ClF3N4O3+·Cl-.H2O (II), its solvated form (sorafenib hydrochloride monohydrate monotetrahydrofuran (Sor·HCl.H2O.THF), C21H17ClF3N4O3+·Cl-.H2O.C4H8O (III)), sorafenib hydrobromide (Sor·HBr), 4-(4-{3-[4-chloro-3-(trifluoro-methyl)phenyl]ureido}phenoxy)-2-(N-methylcarbamoyl) pyridinium hydrobromide, C21H17ClF3N4O3+·Br- (IV) and sorafenib hydrobromide monohydrate (Sor·HBr.H2O), C21H17ClF3N4O3+·Br-.H2O (V) were analysed. Their hydrogen bond systems and topologies were investigated. The results showed the distinct roles of water molecules in stabilizing their crystal structures. Moreover, (II) and (V) were isomorphous crystal structures with the same space group P21/n, and similar unit cell dimensions. The predicted morphologies of these forms based on the BFDH model matched well with experimental morphologies. The energy frameworks showed that (I), and (IV) might have better tabletability than (II) and (V). Moreover, the solubility and dissolution rate data exhibited an improvement in the solubility of these salts compared with the free drug.


Assuntos
Antineoplásicos/química , Ligação de Hidrogênio , Inibidores de Proteínas Quinases/química , Sorafenibe/química , Antineoplásicos/farmacologia , Cristalografia por Raios X , Cristais Líquidos/química , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/farmacologia , Solubilidade , Sorafenibe/farmacologia , Análise Espectral
4.
Int J Mol Sci ; 22(13)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206582

RESUMO

Two new pyrazole derivatives, namely compound 1 and compound 2, have been synthesized, and their biological activity has been evaluated. Monocrystals of the obtained compounds were thoroughly investigated using single-crystal X-ray diffraction analysis, FTIR spectroscopy, and NMR spectroscopy. The results gathered from all three techniques are in good agreement, provide complete information about the structures of 1 and 2, and confirm their high purity. Thermal properties were studied using thermogravimetric analysis; both 1 and 2 are stable at room temperature. In order to better characterize 1 and 2, some physicochemical and biological properties have been evaluated using ADMET analysis. The cytotoxic activity of both compounds was determined using the MTT assay on the A549 cell line in comparison with etoposide. It was determined that compound 2 was effective in the inhibition of human lung adenocarcinoma cell growth and may be a promising compound for the treatment of lung cancer.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Pirazóis/química , Pirazóis/farmacologia , Células A549 , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fenômenos Químicos , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , Análise Espectral , Relação Estrutura-Atividade
5.
Nat Commun ; 12(1): 4195, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234119

RESUMO

SARS-CoV-2 can infect many domestic animals, including dogs. Herein, we show that dog angiotensin-converting enzyme 2 (dACE2) can bind to the SARS-CoV-2 spike (S) protein receptor binding domain (RBD), and that both pseudotyped and authentic SARS-CoV-2 can infect dACE2-expressing cells. We solved the crystal structure of RBD in complex with dACE2 and found that the total number of contact residues, contact atoms, hydrogen bonds and salt bridges at the binding interface in this complex are slightly fewer than those in the complex of the RBD and human ACE2 (hACE2). This result is consistent with the fact that the binding affinity of RBD to dACE2 is lower than that of hACE2. We further show that a few important mutations in the RBD binding interface play a pivotal role in the binding affinity of RBD to both dACE2 and hACE2. Our work reveals a molecular basis for cross-species transmission and potential animal spread of SARS-CoV-2, and provides new clues to block the potential transmission chains of this virus.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , SARS-CoV-2/metabolismo , Enzima de Conversão de Angiotensina 2/química , Animais , Sítios de Ligação , Linhagem Celular , Cricetinae , Cristalografia por Raios X , Cães , Células HeLa , Humanos , Mutação , Ligação Proteica , Domínios Proteicos , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus
6.
Nat Commun ; 12(1): 4236, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244493

RESUMO

The repertoire of peptides presented by major histocompatibility complex class I (MHC-I) molecules on the cell surface is tailored by the ER-resident peptide loading complex (PLC), which contains the exchange catalyst tapasin. Tapasin stabilizes MHC-I molecules and promotes the formation of stable peptide-MHC-I (pMHC-I) complexes that serve as T cell antigens. Exchange of suboptimal by high-affinity ligands is catalyzed by tapasin, but the underlying mechanism is still elusive. Here we analyze the tapasin-induced changes in MHC-I dynamics, and find the catalyst to exploit two essential features of MHC-I. First, tapasin recognizes a conserved allosteric site underneath the α2-1-helix of MHC-I, 'loosening' the MHC-I F-pocket region that accomodates the C-terminus of the peptide. Second, the scoop loop11-20 of tapasin relies on residue L18 to target the MHC-I F-pocket, enabling peptide exchange. Meanwhile, tapasin residue K16 plays an accessory role in catalysis of MHC-I allotypes bearing an acidic F-pocket. Thus, our results provide an explanation for the observed allele-specificity of catalyzed peptide exchange.


Assuntos
Alelos , Apresentação do Antígeno/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Regulação Alostérica , Biocatálise , Cristalografia por Raios X , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Antígenos de Histocompatibilidade Classe I/ultraestrutura , Humanos , Imunoglobulinas/metabolismo , Imunoglobulinas/ultraestrutura , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/isolamento & purificação , Proteínas de Membrana Transportadoras/ultraestrutura , Simulação de Dinâmica Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica em alfa-Hélice , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
7.
Molecules ; 26(12)2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34198604

RESUMO

Two penta-coordinated [Co(MorphBPT)Cl2]; 1 and [Co(PipBPT)Cl2]; 2 complexes with the bis-pyrazolyl-s-triazine pincer ligands MorphBPT and PipBPT were synthesized and characterized. Both MorphBPT and PipBPT act as NNN-tridentate pincer chelates coordinating the Co(II) center with one short Co-N(s-triazine) and two longer Co-N(pyrazole) bonds. The coordination number of Co(II) is five in both complexes, and the geometry around Co(II) ion is a distorted square pyramidal in 1, while 2 shows more distortion. In both complexes, the packing is dominated by Cl…H, C-H…π, and Cl…C (anion-π stacking) interactions in addition to O…H interactions, which are found only in 1. The UV-Vis spectral band at 564 nm was assigned to metal-ligand charge transfer transitions based on TD-DFT calculations. Complexes 1 and 2 showed higher antimicrobial activity compared to the respective free ligand MorphBPT and PipBPT, which were not active. MIC values indicated that 2 had better activity against S. aureus, B. subtilis, and P. vulgaris than 1. DPPH free radical scavenging assay revealed that all the studied compounds showed weak to moderate antioxidant activity where the nature of the substituent at the s-triazine core has a significant impact on the antioxidant activity.


Assuntos
Antibacterianos/farmacologia , Antioxidantes/farmacologia , Cobalto/química , Complexos de Coordenação/síntese química , Cristalografia por Raios X/métodos , Pirazóis/química , Triazinas/química , Antibacterianos/síntese química , Antioxidantes/química , Complexos de Coordenação/farmacologia , Ligantes , Testes de Sensibilidade Microbiana/métodos , Modelos Moleculares
8.
Int J Mol Sci ; 22(12)2021 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-34200865

RESUMO

A novel cytoplasmic dye-decolorizing peroxidase from Dictyostelium discoideum was investigated that oxidizes anthraquinone dyes, lignin model compounds, and general peroxidase substrates such as ABTS efficiently. Unlike related enzymes, an aspartate residue replaces the first glycine of the conserved GXXDG motif in Dictyostelium DyPA. In solution, Dictyostelium DyPA exists as a stable dimer with the side chain of Asp146 contributing to the stabilization of the dimer interface by extending the hydrogen bond network connecting two monomers. To gain mechanistic insights, we solved the Dictyostelium DyPA structures in the absence of substrate as well as in the presence of potassium cyanide and veratryl alcohol to 1.7, 1.85, and 1.6 Å resolution, respectively. The active site of Dictyostelium DyPA has a hexa-coordinated heme iron with a histidine residue at the proximal axial position and either an activated oxygen or CN- molecule at the distal axial position. Asp149 is in an optimal conformation to accept a proton from H2O2 during the formation of compound I. Two potential distal solvent channels and a conserved shallow pocket leading to the heme molecule were found in Dictyostelium DyPA. Further, we identified two substrate-binding pockets per monomer in Dictyostelium DyPA at the dimer interface. Long-range electron transfer pathways associated with a hydrogen-bonding network that connects the substrate-binding sites with the heme moiety are described.


Assuntos
Corantes/química , Dictyostelium/enzimologia , Heme/química , Peróxido de Hidrogênio/química , Peroxidase/química , Peroxidase/metabolismo , Catálise , Domínio Catalítico , Cristalografia por Raios X , Heme/metabolismo , Ligação de Hidrogênio , Oxirredução
9.
Molecules ; 26(11)2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34199417

RESUMO

Blockade of the programmed cell death 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) interaction is currently the focus in the field of cancer immunotherapy, and so far, several monoclonal antibodies (mAbs) have achieved encouraging outcomes in cancer treatment. Despite this achievement, mAbs-based therapies are struggling with limitations including poor tissue and tumor penetration, long half-life time, poor oral bioavailability, and expensive production costs, which prompted a shift towards the development of the small-molecule inhibitors of PD-1/PD-L1 pathways. Even though many small-molecule inhibitors targeting PD-1/PD-L1 interaction have been reported, their development lags behind the corresponding mAb, partly due to the challenges of developing drug-like small molecules. Herein, we report the discovery of a series of novel inhibitors targeting PD-1/PD-L1 interaction via structural simplification strategy by using BMS-1058 as a starting point. Among them, compound A9 stands out as the most promising candidate with excellent PD-L1 inhibitory activity (IC50 = 0.93 nM, LE = 0.43) and high binding affinity to hPD-L1 (KD = 3.64 nM, LE = 0.40). Furthermore, A9 can significantly promote the production of IFN-γ in a dose-dependent manner by rescuing PD-L1 mediated T-cell inhibition in Hep3B/OS-8/hPD-L1 and CD3-positive T cells co-culture assay. Taken together, these results suggest that A9 is a promising inhibitor of PD-1/PD-L1 interaction and is worthy for further study.


Assuntos
Antígeno B7-H1/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Linfócitos T/citologia , Antígeno B7-H1/química , Linhagem Celular , Cristalografia por Raios X , Humanos , Interferon gama/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Cultura Primária de Células , Receptor de Morte Celular Programada 1/química , Ligação Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo
10.
Molecules ; 26(11)2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34199504

RESUMO

The substance class of the well-known Cinchona alkaloids is widened by 6'-Amino-cinchonine and 6'-Amino-cinchonidine, novel compounds which incorporate a primary amino function in the quinolinic ring system. These key intermediates open the field for a range of fruitful chemistry. Here is described a short and direct pathway for the synthesis of triazole containing derivatives of the above-mentioned substances using the [3 + 2] Huisgen cycloaddition. For this purpose, the amines were first converted into the corresponding azides. Based on this, non-substituted and silyl-protected triazoles were synthesized as examples. Furthermore, didehydrated derivatives of quincorine and quincoridine were used as addition partners, resulting in compounds that carry the quinuclidine ring of the cinchona alkaloids at both ends. Some of these compounds were examined radiographically to investigate the position of the quinuclidine ring to the triazole. The solid-state structures of compounds 10, 11 and 28 were determined by X-ray diffraction analyses.


Assuntos
Alcaloides de Cinchona/química , Triazóis/síntese química , Cristalografia por Raios X , Reação de Cicloadição , Modelos Moleculares , Estrutura Molecular , Triazóis/química
11.
Molecules ; 26(12)2021 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-34200764

RESUMO

The cyclooxygenase-2 (COX-2) enzyme is an important target for drug discovery and development of novel anti-inflammatory agents. Selective COX-2 inhibitors have the advantage of reduced side-effects, which result from COX-1 inhibition that is usually observed with nonselective COX inhibitors. In this study, the design and synthesis of a new series of 7-methoxy indolizines as bioisostere indomethacin analogues (5a-e) were carried out and evaluated for COX-2 enzyme inhibition. All the compounds showed activity in micromolar ranges, and the compound diethyl 3-(4-cyanobenzoyl)-7-methoxyindolizine-1,2-dicarboxylate (5a) emerged as a promising COX-2 inhibitor with an IC50 of 5.84 µM, as compared to indomethacin (IC50 = 6.84 µM). The molecular modeling study of indolizines indicated that hydrophobic interactions were the major contribution to COX-2 inhibition. The title compound diethyl 3-(4-bromobenzoyl)-7-methoxyindolizine-1,2-dicarboxylate (5c) was subjected for single-crystal X-ray studies, Hirshfeld surface analysis, and energy framework calculations. The X-ray diffraction analysis showed that the molecule (5c) crystallizes in the monoclinic crystal system with space group P 21/n with a = 12.0497(6)Å, b = 17.8324(10)Å, c = 19.6052(11)Å, α = 90.000°, ß = 100.372(1)°, γ = 90.000°, and V = 4143.8(4)Å3. In addition, with the help of Crystal Explorer software program using the B3LYP/6-31G(d, p) basis set, the theoretical calculation of the interaction and graphical representation of energy value was measured in the form of the energy framework in terms of coulombic, dispersion, and total energy.


Assuntos
Inibidores de Ciclo-Oxigenase 2/química , Indolizinas/química , Anti-Inflamatórios/química , Cristalografia por Raios X/métodos , Ciclo-Oxigenase 2/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Indometacina/química , Relação Estrutura-Atividade
12.
Molecules ; 26(12)2021 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-34204747

RESUMO

Cytochrome P450s (P450) are important enzymes in biology with useful biochemical reactions in, for instance, drug and xenobiotics metabolisms, biotechnology, and health. Recently, the crystal structure of a new member of the CYP116B family has been resolved. This enzyme is a cytochrome P450 (CYP116B46) from Tepidiphilus thermophilus (P450-TT) and has potential for the oxy-functionalization of organic molecules such as fatty acids, terpenes, steroids, and statins. However, it was thought that the opening to its hitherto identified substrate channel was too small to allow organic molecules to enter. To investigate this, we performed molecular dynamics simulations on the enzyme. The results suggest that the crystal structure is not relaxed, possibly due to crystal packing effects, and that its tunnel structure is constrained. In addition, the simulations revealed two key amino acid residues at the mouth of the channel; a glutamyl and an arginyl. The glutamyl's side chain tightens and relaxes the opening to the channel in conjunction with the arginyl's, though the latter's side chain is less dramatically changed after the initial relaxation of its conformations. Additionally, it was observed that the effect of increased temperature did not considerably affect the dynamics of the enzyme fold, including the relative solvent accessibility of the amino acid residues that make up the substrate channel wall even as compared to the changes that occurred at room temperature. Interestingly, the substrate channel became distinguishable as a prominent tunnel that is likely to accommodate small- to medium-sized organic molecules for bioconversions. That is, P450-TT has the ability to pass appropriate organic substrates to its active site through its elaborate substrate channel, and notably, is able to control or gate any molecules at the opening to this channel.


Assuntos
Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Hydrogenophilaceae/metabolismo , Sítios de Ligação/fisiologia , Domínio Catalítico/fisiologia , Cristalografia por Raios X/métodos , Sistema Enzimático do Citocromo P-450/fisiologia , Hydrogenophilaceae/enzimologia , Simulação de Dinâmica Molecular , Oxirredução , Ligação Proteica/fisiologia , Especificidade por Substrato/fisiologia
13.
Molecules ; 26(12)2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208577

RESUMO

Metal-organic frameworks (MOFs) have been rapidly developed for their broad applications in many different chemistry and materials fields. In this work, a multi-dentate building block 5-(4-(tetrazol-5-yl)phenyl)-isophthalic acid (H3L) containing tetrazole and carbolxylate moieties was employed for the synthesis of a two-dimensional (2D) lanthanide MOF [La(HL)(DMF)2(NO3)] (DMF = N,N-dimethylformamide) (1) under solvothermal condition. The fluorescent sensing application of 1 was investigated. 1 exhibits high sensitivity recognition for antibiotic nitrofurantoin (Ksv: 3.0 × 103 M-1 and detection limit: 17.0 µM) and amino acid l-tyrosine (Ksv: 1.4 × 104 M-1 and detection limit: 3.6 µM). This work provides a feasible detection platform of 2D MOFs for highly sensitive discrimination of antibiotics and amino acids.


Assuntos
Elementos da Série dos Lantanídeos/química , Nitrofurantoína/química , Tirosina/química , Antibacterianos/química , Cristalografia por Raios X/métodos , Corantes Fluorescentes/química , Estruturas Metalorgânicas/química , Nitrofurantoína/metabolismo , Tirosina/metabolismo
14.
Science ; 373(6550): 118-121, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34210888

RESUMO

Ethane, the second most abundant hydrocarbon gas in the seafloor, is efficiently oxidized by anaerobic archaea in syntrophy with sulfate-reducing bacteria. Here, we report the 0.99-angstrom-resolution structure of the proposed ethane-activating enzyme and describe the specific traits that distinguish it from methane-generating and -consuming methyl-coenzyme M reductases. The widened catalytic chamber, harboring a dimethylated nickel-containing F430 cofactor, would adapt the chemistry of methyl-coenzyme M reductases for a two-carbon substrate. A sulfur from methionine replaces the oxygen from a canonical glutamine as the nickel lower-axial ligand, a feature conserved in thermophilic ethanotrophs. Specific loop extensions, a four-helix bundle dilatation, and posttranslational methylations result in the formation of a 33-angstrom-long hydrophobic tunnel, which guides the ethane to the buried active site as confirmed with xenon pressurization experiments.


Assuntos
Proteínas Arqueais/química , Etano/química , Methanosarcinales/enzimologia , Oxirredutases/química , Cristalografia por Raios X , Ativação Enzimática , Sequências Hélice-Alça-Hélice , Metilação , Processamento de Proteína Pós-Traducional
15.
Nat Commun ; 12(1): 4181, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234136

RESUMO

Nucleobase and nucleoside analogs (NNA) are widely used as anti-viral and anti-cancer agents, and NNA phosphorylation is essential for the activity of this class of drugs. Recently, diphosphatase NUDT15 was linked to thiopurine metabolism with NUDT15 polymorphism associated with drug toxicity in patients. Profiling NNA drugs, we identify acyclovir (ACV) and ganciclovir (GCV) as two new NNAs metabolized by NUDT15. NUDT15 hydrolyzes ACV and GCV triphosphate metabolites, reducing their effects against cytomegalovirus (CMV) in vitro. Loss of NUDT15 potentiates cytotoxicity of ACV and GCV in host cells. In hematopoietic stem cell transplant patients, the risk of CMV viremia following ACV prophylaxis is associated with NUDT15 genotype (P = 0.015). Donor NUDT15 deficiency is linked to graft failure in patients receiving CMV-seropositive stem cells (P = 0.047). In conclusion, NUDT15 is an important metabolizing enzyme for ACV and GCV, and NUDT15 variation contributes to inter-patient variability in their therapeutic effects.


Assuntos
Aciclovir/farmacologia , Antivirais/farmacologia , Infecções por Citomegalovirus/prevenção & controle , Ganciclovir/análogos & derivados , Pirofosfatases/genética , Aciclovir/uso terapêutico , Adolescente , Adulto , Idoso , Animais , Antibioticoprofilaxia , Antivirais/uso terapêutico , Variação Biológica da População/genética , Linhagem Celular , Criança , Pré-Escolar , Cristalografia por Raios X , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/genética , Citomegalovirus/isolamento & purificação , Infecções por Citomegalovirus/diagnóstico , Infecções por Citomegalovirus/etiologia , Infecções por Citomegalovirus/virologia , DNA Viral/sangue , DNA Viral/isolamento & purificação , Modelos Animais de Doenças , Farmacorresistência Viral , Feminino , Ganciclovir/farmacologia , Ganciclovir/uso terapêutico , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Interações entre Hospedeiro e Microrganismos/genética , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Muromegalovirus/isolamento & purificação , Muromegalovirus/patogenicidade , Variantes Farmacogenômicos , Polimorfismo de Nucleotídeo Único , Pirofosfatases/metabolismo , Pirofosfatases/ultraestrutura , Resultado do Tratamento , Adulto Jovem
16.
Nat Commun ; 12(1): 4226, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244481

RESUMO

RIFIN, a large family of Plasmodium variant surface antigens, plays a crucial role in malaria pathogenesis by mediating immune suppression through activation of inhibitory receptors such as LAIR1, and antibodies with LAIR1 inserts have been identified that bind infected erythrocytes through RIFIN. However, details of RIFIN-mediated LAIR1 recognition and receptor activation have been unclear. Here, we use negative-stain EM to define the architecture of LAIR1-inserted antibodies and determine crystal structures of RIFIN-variable 2 (V2) domain in complex with a LAIR1 domain. These structures reveal the LAIR1-binding region of RIFIN to be hydrophobic and membrane-distal, to exhibit extensive structural diversity, and to interact with RIFIN-V2 in a one-to-one fashion. Through structural and sequence analysis of various LAIR1 constructs, we identify essential elements of RIFIN-binding on LAIR1. Furthermore, a structure-derived LAIR1-binding sequence signature ascertained >20 LAIR1-binding RIFINs, including some from P. falciparum field strains and Plasmodium species infecting gorillas and chimpanzees.


Assuntos
Antígenos de Protozoários/ultraestrutura , Malária Falciparum/imunologia , Proteínas de Membrana/ultraestrutura , Plasmodium falciparum/imunologia , Proteínas de Protozoários/ultraestrutura , Receptores Imunológicos/ultraestrutura , Anticorpos Antiprotozoários/genética , Anticorpos Antiprotozoários/metabolismo , Variação Antigênica/genética , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/metabolismo , Cristalografia por Raios X , Humanos , Malária Falciparum/parasitologia , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Mutação , Plasmodium falciparum/metabolismo , Domínios Proteicos/genética , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo
17.
Nat Commun ; 12(1): 4320, 2021 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-34262046

RESUMO

The rational development of norovirus vaccine candidates requires a deep understanding of the antigenic diversity and mechanisms of neutralization of the virus. Here, we isolate and characterize a panel of broadly cross-reactive naturally occurring human monoclonal IgMs, IgAs and IgGs reactive with human norovirus (HuNoV) genogroup I or II (GI or GII). We note three binding patterns and identify monoclonal antibodies (mAbs) that neutralize at least one GI or GII HuNoV strain when using a histo-blood group antigen (HBGA) blocking assay. The HBGA blocking assay and a virus neutralization assay using human intestinal enteroids reveal that the GII-specific mAb NORO-320, mediates HBGA blocking and neutralization of multiple GII genotypes. The Fab form of NORO-320 neutralizes GII.4 infection more potently than the mAb, however, does not block HBGA binding. The crystal structure of NORO-320 Fab in complex with GII.4 P-domain shows that the antibody recognizes a highly conserved region in the P-domain distant from the HBGA binding site. Dynamic light scattering analysis of GII.4 virus-like particles with mAb NORO-320 shows severe aggregation, suggesting neutralization is by steric hindrance caused by multivalent cross-linking. Aggregation was not observed with the Fab form of NORO-320, suggesting that this clone also has additional inhibitory features.


Assuntos
Anticorpos Antivirais/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , Reações Cruzadas , Norovirus/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Antivirais/química , Anticorpos Antivirais/metabolismo , Variação Antigênica , Sítios de Ligação , Antígenos de Grupos Sanguíneos/metabolismo , Anticorpos Amplamente Neutralizantes/química , Anticorpos Amplamente Neutralizantes/metabolismo , Infecções por Caliciviridae/imunologia , Infecções por Caliciviridae/virologia , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/metabolismo , Cristalografia por Raios X , Mapeamento de Epitopos , Genótipo , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Norovirus/genética , Ligação Proteica , Domínios Proteicos
18.
Nat Commun ; 12(1): 4417, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285212

RESUMO

Endoperoxide-containing natural products are a group of compounds with structurally unique cyclized peroxide moieties. Although numerous endoperoxide-containing compounds have been isolated, the biosynthesis of the endoperoxides remains unclear. NvfI from Aspergillus novofumigatus IBT 16806 is an endoperoxidase that catalyzes the formation of fumigatonoid A in the biosynthesis of novofumigatonin. Here, we describe our structural and functional analyses of NvfI. The structural elucidation and mutagenesis studies indicate that NvfI does not utilize a tyrosyl radical in the reaction, in contrast to other characterized endoperoxidases. Further, the crystallographic analysis reveals significant conformational changes of two loops upon substrate binding, which suggests a dynamic movement of active site during the catalytic cycle. As a result, NvfI installs three oxygen atoms onto a substrate in a single enzyme turnover. Based on these results, we propose a mechanism for the NvfI-catalyzed, unique endoperoxide formation reaction to produce fumigatonoid A.


Assuntos
Aspergillus/enzimologia , Biocatálise , Proteínas Fúngicas/metabolismo , Oxigenases/metabolismo , Peróxidos/metabolismo , Aspergillus/genética , Domínio Catalítico , Cristalografia por Raios X , Compostos Ferrosos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/ultraestrutura , Ácidos Cetoglutáricos/metabolismo , Mutagênese Sítio-Dirigida , Oxirredução , Oxigênio/metabolismo , Oxigenases/genética , Oxigenases/isolamento & purificação , Oxigenases/ultraestrutura , Conformação Proteica em Folha beta , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Terpenos/metabolismo
19.
Int J Mol Sci ; 22(13)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202639

RESUMO

ß-Ketophosphonates with pentalenofurane fragments linked to the keto group were synthesized. The bulky pentalenofurane skeleton is expected to introduce more hindrance in the prostaglandin analogues of type III, greater than that obtained with the bicyclo[3.3.0]oct(a)ene fragments of prostaglandin analogues I and II, to slow down (retard) the inactivation of the prostaglandin analogues by oxidation of 15α-OH to the 15-keto group via the 15-PGDH pathway. Their synthesis was performed by a sequence of three high yield reactions, starting from the pentalenofurane alcohols 2, oxidation of alcohols to acids 3, esterification of acids 3 to methyl esters 4 and reaction of the esters 4 with lithium salt of dimethyl methanephosphonate at low temperature. The secondary compounds 6b and 6c were formed in small amounts in the oxidation reactions of 2b and 2c, and the NMR spectroscopy showed that their structure is that of an ester of the acid with the starting alcohol. Their molecular structures were confirmed by single crystal X-ray determination method for 6c and XRPD powder method for 6b.


Assuntos
Cetonas/química , Organofosfonatos/química , Prostaglandinas Sintéticas/síntese química , Técnicas de Química Sintética , Cristalografia por Raios X , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Prostaglandinas Sintéticas/química , Sesquiterpenos/química
20.
ACS Appl Mater Interfaces ; 13(27): 32226-32241, 2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34210116

RESUMO

The touchstone for bone replacing or anchoring trauma implants, besides resorption, includes functional ankylosis at a fixation point and replacement by viable functional neo-bone tissues. These parameters redefined the concept of "resorbability" as "bioresorbability." Interference screws are the most commonly used resorbable anchoring implants for anterior cruciate ligament (ACL) reconstruction (surgery). Over the years, the bioresorbable screw fixation armamentarium has amplified countless choices, but instability and postimplantation complications have raised concerns about its reliability and efficacy. Owing to this interest, in this work, bioactive glass fiber-reinforced plastic (BGFP) composites with (BGFPnb5) and without (BGFP5) niobicoxide composing multiplexed network modifiers are reported as bioresorbable bone-anchoring substitutes. These synergistically designed composites have a fabricated structure of continuous, unidirectional BG fibers reinforced in an epoxy resin matrix using "melt-drawing and microfabrication" technology. The BGFP microarchitecture is comprised of multiplexed oxide components that influence bioactive response in a distinctive lophelia atoll-like apatite formation. Furthermore, it assists in the proliferation, adherence, and migration of bone marrow-derived mesenchymal stem cells. It also exhibits superior physicochemical characteristics such as surface roughness, hydrophilic exposure, distinctive flexural strength, and bioresorption. Thus, it induces restorative bone osseointegration and osteoconduction and actuates periosteum function. In addition, the BGFP influences the reduction of DH5-α Escherichia coli in suspension culture, demonstrating potential antibacterial efficacy. In conclusion, the BGFP composite therapeutic efficacy demonstrates distinctive material characteristics aiding in bone regeneration and restoration that could serve as a pioneer in orthopedic regenerative medicine.


Assuntos
Substitutos Ósseos/química , Substitutos Ósseos/farmacologia , Vidro/química , Periósteo/efeitos dos fármacos , Plásticos/química , Regeneração Óssea/efeitos dos fármacos , Cristalografia por Raios X , Teste de Materiais , Osseointegração/efeitos dos fármacos , Periósteo/fisiologia , Próteses e Implantes
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