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1.
J Hazard Mater ; 415: 125686, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34088184

RESUMO

The widespread use of antibiotics has led to their ubiquitous presence in water and wastewater and raised concerns about antimicrobial resistance. Clinical antibiotic susceptibility assays have been repurposed to measure removal of antimicrobial activity during water and wastewater treatment processes. The corresponding protocols have mainly employed growth inhibition of Escherichia coli. The present work focused on optimizing bacteria selection to improve the sensitivity of residual antimicrobial activity measurements by broth microdilution assays. Thirteen antibiotics from four classes (i.e., fluoroquinolones, macrolides, sulfonamides, tetracyclines) were investigated against three gram-negative organisms, namely E. coli, Mycoplasma microti, and Pseudomonas fluorescens. The minimum inhibitory concentration (MIC) and half-maximal inhibitory concentration (IC50) were calculated for each antibiotic-bacteria pair. P. fluorescens produces a fluorescent siderophore, pyoverdine, that was used to assess sublethal effects and further enhance the sensitivity of antimicrobial activity measurements. The optimal antibiotic-bacteria pairs were as follows: fluoroquinolone-E. coli (growth inhibition); macrolide- and sulfonamide-M. microti (growth inhibition); and, tetracycline-P. fluorescens (pyoverdine inhibition). Compared to E. coli growth inhibition, the sensitivity of antimicrobial activity analysis was improved by up to 728, 19, and 2.7 times for macrolides (tylosin), sulfonamides (sulfamethoxazole), and tetracyclines (chlortetracycline), facilitating application of these bioassays at environmentally-relevant conditions.


Assuntos
Anti-Infecciosos , Fluoroquinolonas , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Escherichia coli , Fluoroquinolonas/farmacologia , Macrolídeos/farmacologia , Testes de Sensibilidade Microbiana , Mycoplasma , Tetraciclinas/farmacologia
2.
Xenobiotica ; 51(7): 764-770, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34013847

RESUMO

It is important to predict drug-drug interactions via inhibition of intestinal cytochrome P450 3A (CYP3A) which is a determinant of bioavailability of orally administered CYP3A substrates. However, inhibitory effects of macrolide antibiotics on CYP3A-mediated metabolism are not entirely identical between humans and rodents.We investigated the effects of macrolide antibiotics, clarithromycin and erythromycin, on in vitro and in vivo metabolism of triazolam, a CYP3A substrate, in CYP3A-humanised mice generated by using a mouse artificial chromosome vector carrying a human CYP3A gene.Metabolic activities of triazolam were inhibited by macrolide antibiotics in liver and intestine microsomes of CYP3A-humanised mice.The area under the plasma concentration-time curve ratios of 4-hydroxytriazolam to triazolam after oral dosing of triazolam were significantly decreased by multiple administration of macrolide antibiotics. The plasma concentrations ratios of α-hydroxytriazolam and 4-hydroxytriazolam to triazolam in portal blood were significantly decreased by multiple administration of clarithromycin in CYP3A-humanised mice.These results suggest that intestinal CYP3A activity was inhibited by macrolide antibiotics in CYP3A-humanised mice in vitro and in vivo. The plasma concentrations of triazolam and its metabolites in the portal blood of CYP3A-humanised mice would be useful for direct evaluation of intestinal CYP3A-mediated drug-drug interactions.


Assuntos
Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450 , Antibacterianos/farmacologia , Citocromo P-450 CYP3A/genética , Interações Medicamentosas , Humanos , Intestinos , Macrolídeos/farmacologia , Microssomos Hepáticos
3.
Nat Commun ; 12(1): 2803, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33990576

RESUMO

Macrolide antibiotics bind in the nascent peptide exit tunnel of the bacterial ribosome and prevent polymerization of specific amino acid sequences, selectively inhibiting translation of a subset of proteins. Because preventing translation of individual proteins could be beneficial for the treatment of human diseases, we asked whether macrolides, if bound to the eukaryotic ribosome, would retain their context- and protein-specific action. By introducing a single mutation in rRNA, we rendered yeast Saccharomyces cerevisiae cells sensitive to macrolides. Cryo-EM structural analysis showed that the macrolide telithromycin binds in the tunnel of the engineered eukaryotic ribosome. Genome-wide analysis of cellular translation and biochemical studies demonstrated that the drug inhibits eukaryotic translation by preferentially stalling ribosomes at distinct sequence motifs. Context-specific action markedly depends on the macrolide structure. Eliminating macrolide-arrest motifs from a protein renders its translation macrolide-tolerant. Our data illuminate the prospects of adapting macrolides for protein-selective translation inhibition in eukaryotic cells.


Assuntos
Antibacterianos/farmacologia , Macrolídeos/farmacologia , Ribossomos/efeitos dos fármacos , Antibacterianos/química , Sítios de Ligação , Microscopia Crioeletrônica , Células Eucarióticas/efeitos dos fármacos , Células Eucarióticas/metabolismo , Humanos , Macrolídeos/química , Modelos Moleculares , Mutação , Ligação Proteica , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/química , Inibidores da Síntese de Proteínas/farmacologia , RNA Fúngico/genética , RNA Ribossômico/genética , Ribossomos/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Relação Estrutura-Atividade
4.
Nat Commun ; 12(1): 2263, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33859183

RESUMO

Argininosuccinate synthase (ASS1) is a ubiquitous enzyme in mammals that catalyzes the formation of argininosuccinate from citrulline and aspartate. ASS1 genetic deficiency in patients leads to an autosomal recessive urea cycle disorder citrullinemia, while its somatic silence or down-regulation is very common in various human cancers. Here, we show that ASS1 functions as a tumor suppressor in breast cancer, and the pesticide spinosyn A (SPA) and its derivative LM-2I suppress breast tumor cell proliferation and growth by binding to and activating ASS1. The C13-C14 double bond in SPA and LM-2I while the Cys97 (C97) site in ASS1 are critical for the interaction between ASS1 and SPA or LM-2I. SPA and LM-2I treatment results in significant enhancement of ASS1 enzymatic activity in breast cancer cells, particularly in those cancer cells with low ASS1 expression, leading to reduced pyrimidine synthesis and consequently the inhibition of cancer cell proliferation. Thus, our results establish spinosyn A and its derivative LM-2I as potent ASS1 enzymatic activator and tumor inhibitor, which provides a therapeutic avenue for tumors with low ASS1 expression and for those non-tumor diseases caused by down-regulation of ASS1.


Assuntos
Argininossuccinato Sintase/metabolismo , Neoplasias da Mama/tratamento farmacológico , Citrulinemia/tratamento farmacológico , Ativadores de Enzimas/farmacologia , Macrolídeos/farmacologia , Proteínas Supressoras de Tumor/agonistas , Adulto , Idoso , Animais , Argininossuccinato Sintase/genética , Argininossuccinato Sintase/isolamento & purificação , Ácido Aspártico/metabolismo , Mama/patologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citrulina/metabolismo , Citrulinemia/genética , Ativadores de Enzimas/uso terapêutico , Feminino , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Macrolídeos/uso terapêutico , Metabolômica , Camundongos , Pessoa de Meia-Idade , Simulação de Acoplamento Molecular , Mutação , Ligação Proteica , Pirimidinas/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
5.
J Med Microbiol ; 70(4)2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33909552

RESUMO

Introduction. Mycobacterium abscessus complex (MABSC) is an environmental organism and opportunistic pathogen. MABSC pulmonary infections in people with cystic fibrosis are of growing clinical concern. Resistance data guide the use of macrolides and amikacin in MABSC pulmonary disease treatment. MABSC can acquire resistance against macrolides or amikacin via 23S or 16S rRNA gene mutations, respectively.Gap Statement. Current culture-based methods for MABSC detection and antibiotic resistance characterization are typically prolonged, limiting their utility to directly inform treatment or clinical trials. Culture-independent molecular methods may help address this limitation.Aim. To develop real-time PCR assays for characterization of key 23S or 16S rRNA gene mutations associated with constitutive resistance in MABSC.Methodology. We designed two real-time PCR assays to detect the key 23S and 16S rRNA gene mutations. The highly conserved nature of rRNA genes was a major design challenge. To reduce potential cross-reactivity, primers included non-template bases and targeted single-nucleotide polymorphisms unique to MABSC. We applied these assays, as well as a previously developed real-time PCR assay for MABSC detection, to 968 respiratory samples from people with cystic fibrosis. The results from the molecular methods were compared to those for gold standard culture methods and 23S and 16S rRNA gene sequencing.Results.The real-time PCR MABSC detection assay provided a sensitivity of 83.8 % and a specificity of 97.8 % compared to culture. The results from the real-time PCR resistance detection assays were mostly concordant (>77.4 %) with cultured isolate sequencing. The real-time PCR resistance detection assays identified several samples harbouring both resistant and susceptible MABSC, while culture-dependent methods only identified susceptible MABSC in these samples.Conclusion. Using the molecular methods described here, results for health care providers or researchers could be available days or weeks earlier than is currently possible via culture-based antibiotic susceptibility testing.


Assuntos
Amicacina/farmacologia , Antibacterianos/farmacologia , Fibrose Cística/complicações , Macrolídeos/farmacologia , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium abscessus/efeitos dos fármacos , Fibrose Cística/microbiologia , Farmacorresistência Bacteriana , Microbiologia Ambiental , Humanos , Infecções por Mycobacterium não Tuberculosas/complicações , Sistema Respiratório/microbiologia
6.
Mol Cell ; 81(9): 2031-2040.e8, 2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-33909989

RESUMO

Autophagy is a fundamental catabolic process that uses a unique post-translational modification, the conjugation of ATG8 protein to phosphatidylethanolamine (PE). ATG8 lipidation also occurs during non-canonical autophagy, a parallel pathway involving conjugation of ATG8 to single membranes (CASM) at endolysosomal compartments, with key functions in immunity, vision, and neurobiology. It is widely assumed that CASM involves the same conjugation of ATG8 to PE, but this has not been formally tested. Here, we discover that all ATG8s can also undergo alternative lipidation to phosphatidylserine (PS) during CASM, induced pharmacologically, by LC3-associated phagocytosis or influenza A virus infection, in mammalian cells. Importantly, ATG8-PS and ATG8-PE adducts are differentially delipidated by the ATG4 family and bear different cellular dynamics, indicating significant molecular distinctions. These results provide important insights into autophagy signaling, revealing an alternative form of the hallmark ATG8 lipidation event. Furthermore, ATG8-PS provides a specific "molecular signature" for the non-canonical autophagy pathway.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagossomos/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Autofagia , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfatidilserinas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/genética , Autofagossomos/patologia , Família da Proteína 8 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Feminino , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Vírus da Influenza A/patogenicidade , Macrolídeos/farmacologia , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Monensin/farmacologia , Fagocitose , Fosfatidiletanolaminas/metabolismo , Células RAW 264.7 , Transdução de Sinais
7.
Mar Drugs ; 19(4)2021 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-33806230

RESUMO

Macrolides are a significant family of natural products with diverse structures and bioactivities. Considerable effort has been made in recent decades to isolate additional macrolides and characterize their chemical and bioactive properties. The majority of macrolides are obtained from marine organisms, including sponges, marine microorganisms and zooplankton, cnidarians, mollusks, red algae, bryozoans, and tunicates. Sponges, fungi and dinoflagellates are the main producers of macrolides. Marine macrolides possess a wide range of bioactive properties including cytotoxic, antibacterial, antifungal, antimitotic, antiviral, and other activities. Cytotoxicity is their most significant property, highlighting that marine macrolides still encompass many potential antitumor drug leads. This extensive review details the chemical and biological diversity of 505 macrolides derived from marine organisms which have been reported from 1990 to 2020.


Assuntos
Anti-Infecciosos/farmacologia , Antineoplásicos/farmacologia , Organismos Aquáticos/metabolismo , Macrolídeos/farmacologia , Animais , Anti-Infecciosos/isolamento & purificação , Antineoplásicos/isolamento & purificação , Humanos , Macrolídeos/isolamento & purificação , Estrutura Molecular , Relação Estrutura-Atividade
8.
Environ Toxicol ; 36(7): 1316-1325, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33713530

RESUMO

Secondary metabolites in marine organisms exhibit various pharmacological activities against diseases, such as cancer. In this study, the anti-proliferative effect of JBIR-100, a macrolide isolated from Streptomyces sp., was investigated in breast cancer cells. Cell growth was inhibited in response to JBIR-100 treatment concentration- and time-dependently in both MCF-7 and MDA-MB-231 breast cancer cells. JBIR-100 caused apoptosis, as verified by caspase activation and the cleavage of PARP. Western blotting revealed that JBIR-100 modulated the expression of Akt/NF-κB signaling components and Bcl-2 family members. Overexpression of Mcl-1 partially rescued MCF-7 cells from JBIR-100-induced cytotoxicity. In addition, transmission electron microscopy analyses, confocal analysis, and western blot assay indicated that JBIR-100 inhibited autophagy in MCF-7 cells. Exposure to the autophagy inhibitor did not synergize JBIR-100-induced apoptosis. In summary, our results suggested that JBIR-100 may be potentially used for breast cancer therapy.


Assuntos
Neoplasias da Mama , Streptomyces , Apoptose , Autofagia , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Humanos , Células MCF-7 , Macrolídeos/farmacologia
9.
Nat Commun ; 12(1): 1782, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33741963

RESUMO

Pharmacological inhibition of vacuolar-type H+-ATPase (V-ATPase) by its specific inhibitor can abrogate tumor metastasis, prevent autophagy, and reduce cellular signaling responses. Bafilomycin A1, a member of macrolide antibiotics and an autophagy inhibitor, serves as a specific and potent V-ATPases inhibitor. Although there are many V-ATPase structures reported, the molecular basis of specific inhibitors on V-ATPase remains unknown. Here, we report the cryo-EM structure of bafilomycin A1 bound intact bovine V-ATPase at an overall resolution of 3.6-Å. The structure reveals six bafilomycin A1 molecules bound to the c-ring. One bafilomycin A1 molecule engages with two c subunits and disrupts the interactions between the c-ring and subunit a, thereby preventing proton translocation. Structural and sequence analyses demonstrate that the bafilomycin A1-binding residues are conserved in yeast and mammalian species and the 7'-hydroxyl group of bafilomycin A1 acts as a unique feature recognized by subunit c.


Assuntos
Macrolídeos/farmacologia , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Sítios de Ligação , Biocatálise/efeitos dos fármacos , Bovinos , Microscopia Crioeletrônica , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Macrolídeos/química , Macrolídeos/metabolismo , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Domínios Proteicos , Homologia de Sequência de Aminoácidos , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/ultraestrutura
10.
Nat Commun ; 12(1): 1732, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33741980

RESUMO

Macrolides are a class of antibiotics widely used in both medicine and agriculture. Unsurprisingly, as a consequence of their exensive usage a plethora of resistance mechanisms have been encountered in pathogenic bacteria. One of these resistance mechanisms entails the enzymatic cleavage of the macrolides' macrolactone ring by erythromycin esterases (Eres). The most frequently identified Ere enzyme is EreA, which confers resistance to the majority of clinically used macrolides. Despite the role Eres play in macrolide resistance, research into this family enzymes has been sparse. Here, we report the first three-dimensional structures of an erythromycin esterase, EreC. EreC is an extremely close homologue of EreA, displaying more than 90% sequence identity. Two structures of this enzyme, in conjunction with in silico flexible docking studies and previously reported mutagenesis data allowed for the proposal of a detailed catalytic mechanism for the Ere family of enzymes, labeling them as metal-independent hydrolases. Also presented are substrate spectrum assays for different members of the Ere family. The results from these assays together with an examination of residue conservation for the macrolide binding site in Eres, suggests two distinct active site archetypes within the Ere enzyme family.


Assuntos
Antibacterianos/química , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Esterases/química , Esterases/genética , Macrolídeos/química , Antibacterianos/farmacologia , Bactérias/enzimologia , Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Eritromicina/química , Genes Bacterianos , Macrolídeos/farmacologia , Simulação de Acoplamento Molecular , Conformação Proteica , Difração de Raios X
11.
Nat Commun ; 12(1): 1864, 2021 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-33767144

RESUMO

Extracellular vesicles (EVs), including exosomes, are thought to mediate intercellular communication through the transfer of cargoes from donor to acceptor cells. Occurrence of EV-content delivery within acceptor cells has not been unambiguously demonstrated, let alone quantified, and remains debated. Here, we developed a cell-based assay in which EVs containing luciferase- or fluorescent-protein tagged cytosolic cargoes are loaded on unlabeled acceptor cells. Results from dose-responses, kinetics, and temperature-block experiments suggest that EV uptake is a low yield process (~1% spontaneous rate at 1 h). Further characterization of this limited EV uptake, through fractionation of membranes and cytosol, revealed cytosolic release (~30% of the uptaken EVs) in acceptor cells. This release is inhibited by bafilomycin A1 and overexpression of IFITM proteins, which prevent virus entry and fusion. Our results show that EV content release requires endosomal acidification and suggest the involvement of membrane fusion.


Assuntos
Antígenos de Diferenciação/metabolismo , Transporte Biológico/fisiologia , Comunicação Celular/fisiologia , Vesículas Extracelulares/metabolismo , Linhagem Celular Tumoral , Citosol/metabolismo , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes/metabolismo , Células HEK293 , Células HeLa , Humanos , Luciferases/metabolismo , Macrolídeos/farmacologia , Fusão de Membrana/fisiologia
12.
Oxid Med Cell Longev ; 2021: 6675264, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33728026

RESUMO

Acute lung injury (ALI) is a serious respiratory syndrome characterized with uncontrolled inflammatory response. Oxyberberine has strong potential for clinical usage since it showed strong anti-inflammatory, antifungal, and antiarrhythmic effects in various diseases. In the present study, we evaluated whether oxyberberine can inhibit lipopolysaccharide- (LPS-) induced ALI in vivo and further evaluated the possible involvement of mitophagy in vitro by using A549 cells, a human lung epithelial cell line. Our in vivo study shows that oxyberberine significantly inhibited LPS-induced lung pathological injury and lung edema, as indicated by the changes in lung wet/dry ratio and total protein levels in the BALF in mice. Moreover, oxyberberine inhibited inflammation, as indicated by the changes of neutrophil accumulation and production of proinflammatory cytokines including tumor necrosis factor α (TNF-α), interleukin 1ß (IL-1ß), and IL-6 in both the lung and bronchoalveolar lavage fluid (BALF) in ALI mice. Our in vitro study shows that LPS significantly decreased the protein level of mitochondrial proteins, including cytochrome c oxidase subunit IV (COX IV), p62, and mitofusin-2 (Mfn2) in A549 cells. In addition, LPS induced significant Parkin1 translocation from cytoplasm to mitochondria. These changes were significantly inhibited by oxyberberine. Notably, the inhibitory effect of oxyberberine was almost totally lost in the presence of lysosome fusion inhibitor bafilomycin A1 (Baf), a mitophagy inhibitor. In conclusion, the present study demonstrated that oxyberberine alleviated LPS-induced inflammation in ALI via inhibition of Parkin-mediated mitophagy.


Assuntos
Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/prevenção & controle , Berberina/uso terapêutico , Mitofagia , Células A549 , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/patologia , Animais , Apoptose/efeitos dos fármacos , Berberina/farmacologia , Líquido da Lavagem Broncoalveolar , Edema/patologia , Humanos , Inflamação/patologia , Lipopolissacarídeos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Macrolídeos/farmacologia , Masculino , Camundongos Endogâmicos BALB C , Mitofagia/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/patologia , Espécies Reativas de Oxigênio/metabolismo
13.
Appl Environ Microbiol ; 87(10)2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33637575

RESUMO

A group of polyene macrolides mainly composed of two constituents was isolated from the fermentation broth of Streptomyces roseoflavus Men-myco-93-63, which was isolated from soil where potato scabs were repressed naturally. One of these macrolides was roflamycoin, which was first reported in 1968, and the other was a novel compound named Men-myco-A, which had one methylene unit more than roflamycoin. Together, they were designated RM. This group of antibiotics exhibited broad-spectrum antifungal activities in vitro against 17 plant-pathogenic fungi, with 50% effective concentrations (EC50) of 2.05 to 7.09 µg/ml and 90% effective concentrations (EC90) of 4.32 to 54.45 µg/ml, which indicates their potential use in plant disease control. Furthermore, their biosynthetic gene cluster was identified, and the associated biosynthetic assembly line was proposed based on a module and domain analysis of polyketide synthases (PKSs), supported by findings from gene inactivation experiments.IMPORTANCE Streptomyces roseoflavus Men-myco-93-63 is a biocontrol strain that has been studied in our laboratory for many years and exhibits a good inhibitory effect in many crop diseases. Therefore, the identification of antimicrobial metabolites is necessary and our main objective. In this work, chemical, bioinformatic, and molecular biological methods were combined to identify the structures and biosynthesis of the active metabolites. This work provides a new alternative agent for the biological control of plant diseases and is helpful for improving both the properties and yield of the antibiotics via genetic engineering.


Assuntos
Agentes de Controle Biológico , Macrolídeos/metabolismo , Polienos/metabolismo , Streptomyces/metabolismo , Ascomicetos/efeitos dos fármacos , Ascomicetos/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Vias Biossintéticas/genética , Macrolídeos/farmacologia , Família Multigênica , Micélio/efeitos dos fármacos , Micélio/crescimento & desenvolvimento , Doenças das Plantas/prevenção & controle , Polienos/farmacologia , Streptomyces/genética
14.
J Nat Prod ; 84(2): 537-543, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33631936

RESUMO

A new bafilomycin derivative (1) and another seven known bafilomycins (2-8) were isolated from feces-derived Streptomyces sp. HTL16. The structure of 1 was elucidated by 1D and 2D NMR spectroscopic analysis. Biological testing demonstrated that these bafilomycins exhibited potent antiviral activities against the influenza A and SARS-CoV-2 viruses, with IC50 values in the nanomolar range, by inhibiting the activity of endosomal ATP-driven proton pumps.


Assuntos
Antivirais/farmacologia , Fezes/microbiologia , Macrolídeos/farmacologia , ATPases Translocadoras de Prótons/antagonistas & inibidores , Streptomyces/metabolismo , Animais , Cães , Vírus da Influenza A/efeitos dos fármacos , Células Madin Darby de Rim Canino , SARS-CoV-2/efeitos dos fármacos
15.
Virol J ; 18(1): 46, 2021 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-33639976

RESUMO

BACKGROUND: Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2 and broke out as a global pandemic in late 2019. The acidic pH environment of endosomes is believed to be essential for SARS-CoV-2 to be able to enter cells and begin replication. However, the clinical use of endosomal acidification inhibitors, typically chloroquine, has been controversial with this respect. METHODS: In this study, RT-qPCR method was used to detect the SARS-CoV-2N gene to evaluate viral replication. The CCK-8 assay was also used to evaluate the cytotoxic effect of SARS-CoV-2. In situ hybridization was used to examine the distribution of the SARS-CoV-2 gene in lung tissues. Hematoxylin and eosin staining was also used to evaluate virus-associated pathological changes in lung tissues. RESULTS: In this study, analysis showed that endosomal acidification inhibitors, including chloroquine, bafilomycin A1 and NH4CL, significantly reduced the viral yields of SARS-CoV-2 in Vero E6, Huh-7 and 293T-ACE2 cells. Chloroquine and bafilomycin A1 also improved the viability and proliferation of Vero E6 cells after SARS-CoV-2 infection. Moreover, in the hACE2 transgenic mice model of SARS-CoV-2 infection, chloroquine and bafilomycin A1 reduced viral replication in lung tissues and alleviated viral pneumonia with reduced inflammatory exudation and infiltration in peribronchiolar and perivascular tissues, as well as improved structures of alveolar septum and pulmonary alveoli. CONCLUSIONS: Our research investigated the antiviral effects of endosomal acidification inhibitors against SARS-CoV-2 in several infection models and provides an experimental basis for further mechanistic studies and drug development.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , COVID-19/virologia , Endossomos/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Replicação Viral/efeitos dos fármacos , Cloreto de Amônio/farmacologia , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , COVID-19/metabolismo , COVID-19/patologia , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Cloroquina/farmacologia , Endossomos/metabolismo , Feminino , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Pulmão/patologia , Macrolídeos/farmacologia , Camundongos , Camundongos Transgênicos , Distribuição Aleatória , SARS-CoV-2/genética , Células Vero
16.
J Med Microbiol ; 70(3)2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33570485

RESUMO

Introduction. Mycobacterium abscessus complex (MABC) is an infectious agent associated with macrolide resistance and treatment failure.Hypothesis/Gap Statement. Despite drug-susceptibility testing for MABC isolates including clarithromycin (CAM), long-term treatment with azithromycin (AZM) for MABC disease is recommended.Aim. We compared phenotypic and genotypic resistance to AZM and CAM in clinical isolates and evaluated the accumulation of intrinsic macrolide resistance (AIM) and morphological changes by macrolides exposure.Methodology. Forty-nine isolates were characterized regarding erm(41) sequevars. Sequencing data were compared to the nucleotide sequence of rrl and whiB7. The AIM MIC was performed in three reference strains and 15 isolates were randomized [each set of five isolates with M. abscessus subsp. abscessus (MAA) T28, MAA C28 and subsp. massiliense (MAM)].Results. The 49 isolates were distributed as 24 MAA T28, 5 MAA C28 and 20 MAM. The MIC50 values to CAM at day 3 in MAA T28, C28 and MAM were 1, 0.12 and 0.12 µg ml-1, while those at day 14 were 32, 0.5 and 0.12 µg ml-1, respectively. The AZM-MIC50 values at day 3 of the above isolates were 4, 0.25 and 0.5 µg ml-1, while those at day 14 were >64, 0.5 and 0.5 µg ml-1, respectively. Neither mutations in rrl of MAA T28 with acquired resistance nor deletions in whiB7 of MAA T28 without inducible resistance were observed . For AIM MIC, MAA T28 showed that the time-to-detection of AZM resistance was significantly faster over that of CAM (P<0.05). Morphological changes were not determined in all isolates.Conclusion. Our findings did not support the suggestion for the preferential use of AZM for, at least, MAA T28 disease due to the high-level MIC value and the increased AIM. The long duration of AZM-based treatment eventually may favour the emergence of isolates with a high-level of intrinsic resistance.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Macrolídeos/farmacologia , Mycobacterium abscessus/isolamento & purificação , Azitromicina/farmacologia , Azitromicina/uso terapêutico , Proteínas de Bactérias/genética , Claritromicina/farmacologia , Claritromicina/uso terapêutico , Genótipo , Humanos , Testes de Sensibilidade Microbiana , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium abscessus/efeitos dos fármacos , Mycobacterium abscessus/genética , Fenótipo
17.
PLoS Negl Trop Dis ; 15(2): e0009064, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33600426

RESUMO

Several issues have been identified with the current programs for the elimination of onchocerciasis that target only transmission by using mass drug administration (MDA) of the drug ivermectin. Alternative and/or complementary treatment regimens as part of a more comprehensive strategy to eliminate onchocerciasis are needed. We posit that the addition of "prophylactic" drugs or therapeutic drugs that can be utilized in a prophylactic strategy to the toolbox of present microfilaricidal drugs and/or future macrofilaricidal treatment regimens will not only improve the chances of meeting the elimination goals but may hasten the time to elimination and also will support achieving a sustained elimination of onchocerciasis. These "prophylactic" drugs will target the infective third- (L3) and fourth-stage (L4) larvae of Onchocerca volvulus and consequently prevent the establishment of new infections not only in uninfected individuals but also in already infected individuals and thus reduce the overall adult worm burden and transmission. Importantly, an effective prophylactic treatment regimen can utilize drugs that are already part of the onchocerciasis elimination program (ivermectin), those being considered for MDA (moxidectin), and/or the potential macrofilaricidal drugs (oxfendazole and emodepside) currently under clinical development. Prophylaxis of onchocerciasis is not a new concept. We present new data showing that these drugs can inhibit L3 molting and/or inhibit motility of L4 at IC50 and IC90 that are covered by the concentration of these drugs in plasma based on the corresponding pharmacological profiles obtained in human clinical trials when these drugs were tested using various doses for the therapeutic treatments of various helminth infections.


Assuntos
Filaricidas/farmacologia , Leucócitos Mononucleares/parasitologia , Onchocerca volvulus/efeitos dos fármacos , Animais , Benzimidazóis/farmacologia , Depsipeptídeos/farmacologia , Humanos , Ivermectina/farmacologia , Larva/efeitos dos fármacos , Macrolídeos/farmacologia , Onchocerca volvulus/crescimento & desenvolvimento , Oncocercose/tratamento farmacológico , Oncocercose/prevenção & controle
18.
Curr Pharm Des ; 27(9): 1202-1210, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33550966

RESUMO

BACKGROUND: The spike (S) glycoprotein of SARS corona virus (SARS-CoV-2) and human Angiotensin- converting enzyme 2 (ACE2), are both considered the key factors for the initiation of virus infection. The present work is an effort for computational target to block the spike proteins (S) and ACE2 receptor proteins with Macrolide antibiotics like Azithromycin, (AZM), Clarithromycin (CLAM) and Erythromycin (ERY) along with RNA-dependent RNA polymerase (RdRp). METHODS: Three-dimensional structure of the SARS-CoV-2RdRp was built by the SWISS-MODEL server, the generated structure showed 96.35% identity to the available structure of SARS-Coronavirus NSP12 (6NUR), for model validity, we utilized the SWISS-model server quality parameters and Ramachandran plots. RESULTS: These compounds were able to block the residues (Arg553, Arg555, and Ala558) surrounding the deep grove catalytic site (Val557) of RdRp and thus plays an important role in tight blocking of enzyme active site. Reference drug Remdesivir was used to compare the docking score of antibiotics with RdRp. Docking value exhibited good binding energy (-7.7 up to -8.2 kcal/mol) with RdRp, indicating their potential as a potent RdRp inhibitor. Interaction of CLAM and ERY presented low binding energy (-6.8 and -6.6) with the ACE2 receptor. At the same time, CLAM exhibited a good binding affinity of -6.4 kcal/mol, making it an excellent tool to block the attachment of spike protein to ACE2 receptors. Macrolides not only affected the attachment to ACE2 but also blocked the spike proteins further, consequently inhibiting the internalization in the host cell. Three Alkyl bonds between Arg555, Ala558, and Met542 by CLAM and two Alkyl bonds of Arg624 and Lys621 by ERY plays an important role for RdRp inactivation, that can prevent the rise of newly budded progeny virus. These macrolides interacted with the main protease protein in the pocket responsible for the dimerization and catalytic function of this protein. The interaction occurred with residue Glu166, along with the catalytic residues (Tyr343, and His235) of Endoribonuclease (NSP15) protein. CONCLUSION: The present study gives three-way options either by blocking S proteins or ACE2 receptor proteins or inhibiting RdRp to counter any effect of COVID-19 by macrolide and could be useful in the treatment of COVID-19 till some better option available.


Assuntos
COVID-19 , Antibacterianos/farmacologia , Antivirais , Humanos , Macrolídeos/farmacologia , Ligação Proteica , SARS-CoV-2
19.
Emerg Infect Dis ; 27(2): 529-537, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33496218

RESUMO

Multidrug resistance has been detected in the animal and zoonotic human pathogen Rhodococcus equi after mass macrolide/rifampin antibioprophylaxis in endemically affected equine farms in the United States. Multidrug-resistant (MDR) R. equi emerged upon acquisition of pRERm46, a conjugative plasmid conferring resistance to macrolides, lincosamides, streptogramins, and, as we describe, tetracycline. Phylogenomic analyses indicate that the increasing prevalence of MDR R. equi since it was first documented in 2002 is caused by a clone, R. equi 2287, attributable to coselection of pRErm46 with a chromosomal rpoBS531F mutation driven by macrolide/rifampin therapy. pRErm46 spillover to other R. equi genotypes has given rise to a novel MDR clone, G2016, associated with a distinct rpoBS531Y mutation. Our findings illustrate that overuse of antimicrobial prophylaxis in animals can generate MDR pathogens with zoonotic potential. MDR R. equi and pRErm46-mediated resistance are currently disseminating in the United States and are likely to spread internationally through horse movements.


Assuntos
Infecções por Actinomycetales , Doenças dos Cavalos , Rhodococcus equi , Rhodococcus , Infecções por Actinomycetales/tratamento farmacológico , Infecções por Actinomycetales/epidemiologia , Infecções por Actinomycetales/veterinária , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana , Doenças dos Cavalos/epidemiologia , Cavalos , Macrolídeos/farmacologia , Rhodococcus equi/genética , Estados Unidos/epidemiologia
20.
Carbohydr Polym ; 255: 117484, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33436244

RESUMO

Wound dressing composed of chitosan, based crosslinked gelatin/ polyvinyl pyrrolidone, embedded silver nanoparticles were fabricated using solution casting method. The membrane was characterized by FTIR, SEM and TGA. Glutaraldehyde (0.5 %) was used for the crosslinking of membrane components and associated with 7-folds boosted mechanical performance, 28 % more hydrolytic stability, 3-folds thickness reduction and morphological roughness. Silver nanoparticles were characterized by UV-vis, XRD and TEM for an average size of 9.9 nm. The membrane with higher concentration of silver nanoparticles showed maximum antibacterial activity against human pathogenic bacteria; and the measured inhibition zones ranged from 1.5 to 3 cm. The activity of the particles ranged from severe to complete reduction in Penicillin, Erythromycin and Macrolide family's resistance genes expression such as ß-Lactamase, mecA and erm. This developed membrane can serve as promising and cost-effective system against severe diabetic and burn wound infections.


Assuntos
Antibacterianos/farmacologia , Bandagens , Quitosana/química , Citrullus colocynthis/química , Gelatina/química , Povidona/química , Prata/farmacologia , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Eritromicina/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Macrolídeos/farmacologia , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Metiltransferases/genética , Metiltransferases/metabolismo , Testes de Sensibilidade Microbiana , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Penicilinas/farmacologia , Cultura Primária de Células , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Salmonella typhi/efeitos dos fármacos , Salmonella typhi/crescimento & desenvolvimento , Prata/química , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , beta-Lactamases/genética , beta-Lactamases/metabolismo
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