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1.
mBio ; 11(5)2020 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-33082259

RESUMO

The emergence of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), the etiological agent of the 2019 coronavirus disease (COVID-19), has erupted into a global pandemic that has led to tens of millions of infections and hundreds of thousands of deaths worldwide. The development of therapeutics to treat infection or as prophylactics to halt viral transmission and spread is urgently needed. SARS-CoV-2 relies on structural rearrangements within a spike (S) glycoprotein to mediate fusion of the viral and host cell membranes. Here, we describe the development of a lipopeptide that is derived from the C-terminal heptad repeat (HRC) domain of SARS-CoV-2 S that potently inhibits infection by SARS-CoV-2. The lipopeptide inhibits cell-cell fusion mediated by SARS-CoV-2 S and blocks infection by live SARS-CoV-2 in Vero E6 cell monolayers more effectively than previously described lipopeptides. The SARS-CoV-2 lipopeptide exhibits broad-spectrum activity by inhibiting cell-cell fusion mediated by SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV) and blocking infection by live MERS-CoV in cell monolayers. We also show that the SARS-CoV-2 HRC-derived lipopeptide potently blocks the spread of SARS-CoV-2 in human airway epithelial (HAE) cultures, an ex vivo model designed to mimic respiratory viral propagation in humans. While viral spread of SARS-CoV-2 infection was widespread in untreated airways, those treated with SARS-CoV-2 HRC lipopeptide showed no detectable evidence of viral spread. These data provide a framework for the development of peptide therapeutics for the treatment of or prophylaxis against SARS-CoV-2 as well as other coronaviruses.IMPORTANCE SARS-CoV-2, the causative agent of COVID-19, continues to spread globally, placing strain on health care systems and resulting in rapidly increasing numbers of cases and mortalities. Despite the growing need for medical intervention, no FDA-approved vaccines are yet available, and treatment has been limited to supportive therapy for the alleviation of symptoms. Entry inhibitors could fill the important role of preventing initial infection and preventing spread. Here, we describe the design, synthesis, and evaluation of a lipopeptide that is derived from the HRC domain of the SARS-CoV-2 S glycoprotein that potently inhibits fusion mediated by SARS-CoV-2 S glycoprotein and blocks infection by live SARS-CoV-2 in both cell monolayers (in vitro) and human airway tissues (ex vivo). Our results highlight the SARS-CoV-2 HRC-derived lipopeptide as a promising therapeutic candidate for SARS-CoV-2 infections.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Lipopeptídeos/farmacologia , Glicoproteína da Espícula de Coronavírus/química , Internalização do Vírus/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Antivirais/química , Betacoronavirus/química , Betacoronavirus/fisiologia , Chlorocebus aethiops , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/transmissão , Células HEK293 , Humanos , Lipopeptídeos/química , Fusão de Membrana/efeitos dos fármacos , Coronavírus da Síndrome Respiratória do Oriente Médio/química , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Pneumonia Viral/transmissão , Domínios Proteicos , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/virologia , Vírus da SARS/química , Vírus da SARS/efeitos dos fármacos , Vírus da SARS/fisiologia , Células Vero
2.
Anticancer Res ; 40(10): 5361-5369, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32988855

RESUMO

BACKGROUND/AIM: The aim of this study was to evaluate the role of toll-like receptor 2 (TLR2) in the proliferation of human lung cancer cells and identify the signaling pathway that mediates this effect. MATERIALS AND METHODS: Adenocarcinoma (A549 and H1650) and adenosquamous (H125) cells were treated with increasing doses of Pam3CSK4, a TLR2 agonist. Cell proliferation and NF-ĸB activation were evaluated. NF-ĸB was inhibited prior to treatment with Pam3CSK4 and proliferation was assessed. RESULTS: TLR2 expression was significantly higher in A549 and H1650 cells compared to H125 cells (p<0.001). TLR2 stimulation induced proliferation in adenocarcinoma cells only and led to a corresponding increase in NF-ĸB activity (p<0.05). Inhibition of NF-ĸB prior to treatment with Pam3CSK4 attenuated this proliferative response. CONCLUSION: TLR2 activation induced proliferation of lung adenocarcinoma cells through activation of NF-ĸB. Thus, the TLR2 signaling pathway may be a potential therapeutic target in lung adenocarcinoma.


Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Lipopeptídeos/farmacologia , Receptor 2 Toll-Like/genética , Células A549 , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , NF-kappa B/genética , Receptor 2 Toll-Like/agonistas
3.
Appl Environ Microbiol ; 86(21)2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32859591

RESUMO

In the present study, a deep-sea bacterial strain designated Bacillus sp. strain wsm-1 was screened and found to exhibit strong antifungal activity against many plant-pathogenic fungi, and corresponding antifungal agents were thereby purified and determined by tandem mass spectrometry to be two cyclic lipopeptide homologs. These homologs, which were different from any previously reported lipopeptides, were identified to possess identical amino acid sequences of ß-amino fatty acid-Asn-Ser-Asn-Pro-Tyr-Asn-Gln and deduced as two novel lipopeptides designated C14 iturin W and C15 iturin W. Electron microscopy observation indicated that both iturin W homologs caused obvious morphological changes and serious disruption of plasma membrane toward fungal cells, while C15 iturin W exhibited more serious cell damages than C14 iturin W did, which was well consistent with the results of the antifungal activity assays. To improve the yield and antifungal activity of iturin W, the effects of different carbon and nitrogen sources and amino acids on production of C14 iturin W and C15 iturin W were investigated. The results indicated that supplements of most of the detected carbon and nitrogen sources could increase the yield of C14 iturin W, but inhibit the yield of C15 iturin W, while supplements of tryptone and most of the detected amino acids could increase the yield of both C14 iturin W and C15 iturin W.IMPORTANCE Plant disease caused by pathogenic fungi is one of the most devastating diseases, which affects the food safety of the whole world to a great extent. Biological control of plant diseases by microbial natural products is more desirable than traditional chemical control. In this study, we discovered a novel lipopeptide, iturin W, with promising prospects in biological control of plant diseases. Moreover, the effects of different carbon and nitrogen sources and amino acids on production of C14 iturin W and C15 iturin W would provide a reasonable basis for the optimization of the fermentation process of lipopeptides. Notably, the structure of iturin W was different from that of any previously reported lipopeptide, suggesting that deep-sea microorganisms might produce many novel natural products and have significant potential in the development of biological products in the future.


Assuntos
Antifúngicos/farmacologia , Proteínas de Bactérias/farmacologia , Fungos/efeitos dos fármacos , Lipopeptídeos/farmacologia , Peptídeos Cíclicos/fisiologia , Alternaria/efeitos dos fármacos , Antifúngicos/química , Bacillus , Proteínas de Bactérias/química , Colletotrichum/efeitos dos fármacos , Fungicidas Industriais/química , Fungicidas Industriais/farmacologia , Fusarium/efeitos dos fármacos , Lipopeptídeos/química , Magnaporthe/efeitos dos fármacos , Peptídeos Cíclicos/química , Análise de Sequência de Proteína
4.
Nat Commun ; 11(1): 3688, 2020 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-32703948

RESUMO

Zeta inhibitory peptide (ZIP), a PKMζ inhibitor, is widely used to interfere with the maintenance of acquired memories. ZIP is able to erase memory even in the absence of PKMζ, via an unknown mechanism. We found that ZIP induces redistribution of the AMPARGluA1 in HEK293 cells and primary cortical neurons, and decreases AMPAR-mediated currents in the nucleus accumbens (NAc). These effects were mimicked by free arginine or by a modified ZIP in which all but the arginine residues were replaced by alanine. Redistribution was blocked by a peptidase-resistant version of ZIP and by treatment with the nitric oxide (NO)-synthase inhibitor L-NAME. ZIP increased GluA1-S831 phosphorylation and ZIP-induced redistribution was blocked by nitrosyl-mutant GluA1-C875S or serine-mutant GluA1-S831A. Introducing the cleavable arginine-alanine peptide into the NAc attenuated expression of cocaine-conditioned reward. Together, these results suggest that ZIP may act as an arginine donor, facilitating NO-dependent downregulation of AMPARs, thereby attenuating learning and memory.


Assuntos
Peptídeos Penetradores de Células/farmacologia , Condicionamento Psicológico/efeitos dos fármacos , Lipopeptídeos/farmacologia , Memória de Longo Prazo/efeitos dos fármacos , Óxido Nítrico/metabolismo , Receptores de AMPA/metabolismo , Animais , Cocaína/administração & dosagem , Regulação para Baixo , Endocitose/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Células HEK293 , Humanos , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Camundongos , Modelos Animais , NG-Nitroarginina Metil Éster/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Óxido Nítrico/antagonistas & inibidores , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/fisiologia , Fosforilação , Cultura Primária de Células , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Ratos , Receptores de AMPA/genética , Recompensa , Técnicas Estereotáxicas
6.
Pharmacol Res ; 157: 104820, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32360484

RESUMO

The Coronavirus Disease 2019 (COVID-19) pandemic has become a huge threaten to global health, which raise urgent demand of developing efficient therapeutic strategy. The aim of the present study is to dissect the chemical composition and the pharmacological mechanism of Qingfei Paidu Decoction (QFPD), a clinically used Chinese medicine for treating COVID-19 patients in China. Through comprehensive analysis by liquid chromatography coupled with high resolution mass spectrometry (MS), a total of 129 compounds of QFPD were putatively identified. We also constructed molecular networking of mass spectrometry data to classify these compounds into 14 main clusters, in which exhibited specific patterns of flavonoids (45 %), glycosides (15 %), carboxylic acids (10 %), and saponins (5 %). The target network model of QFPD, established by predicting and collecting the targets of identified compounds, indicated a pivotal role of Ma Xing Shi Gan Decoction (MXSG) in the therapeutic efficacy of QFPD. Supportively, through transcriptomic analysis of gene expression after MXSG administration in rat model of LPS-induced pneumonia, the thrombin and Toll-like receptor (TLR) signaling pathway were suggested to be essential pathways for MXSG mediated anti-inflammatory effects. Besides, changes in content of major compounds in MXSG during decoction were found by the chemical analysis. We also validate that one major compound in MXSG, i.e. glycyrrhizic acid, inhibited TLR agonists induced IL-6 production in macrophage. In conclusion, the integration of in silico and experimental results indicated that the therapeutic effects of QFPD against COVID-19 may be attributed to the anti-inflammatory effects of MXSG, which supports the rationality of the compatibility of TCM.


Assuntos
Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Medicamentos de Ervas Chinesas/análise , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , Pneumonia Viral/tratamento farmacológico , Animais , Anti-Inflamatórios/análise , Anti-Inflamatórios/farmacologia , Células Cultivadas , Simulação por Computador , Infecções por Coronavirus/genética , Expressão Gênica/efeitos dos fármacos , Ácido Glicirrízico/farmacologia , Humanos , Interleucina-6/metabolismo , Lipopeptídeos/antagonistas & inibidores , Lipopeptídeos/farmacologia , Lipopolissacarídeos , Masculino , Pandemias , Pneumonia/induzido quimicamente , Pneumonia/metabolismo , Pneumonia Viral/genética , Ratos , Transdução de Sinais/efeitos dos fármacos , Trombina/metabolismo , Receptores Toll-Like/metabolismo
7.
J Virol ; 94(14)2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32376627

RESUMO

The 2019 coronavirus disease (COVID-19), caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has posed serious threats to global public health and economic and social stabilities, calling for the prompt development of therapeutics and prophylactics. In this study, we first verified that SARS-CoV-2 uses human angiotensin-converting enzyme 2 (ACE2) as a cell receptor and that its spike (S) protein mediates high membrane fusion activity. The heptad repeat 1 (HR1) sequence in the S2 fusion protein of SARS-CoV-2 possesses markedly increased α-helicity and thermostability, as well as a higher binding affinity with its corresponding heptad repeat 2 (HR2) site, than the HR1 sequence in S2 of severe acute respiratory syndrome coronavirus (SARS-CoV). Then, we designed an HR2 sequence-based lipopeptide fusion inhibitor, termed IPB02, which showed highly potent activities in inhibiting SARS-CoV-2 S protein-mediated cell-cell fusion and pseudovirus transduction. IPB02 also inhibited the SARS-CoV pseudovirus efficiently. Moreover, the structure-activity relationship (SAR) of IPB02 was characterized with a panel of truncated lipopeptides, revealing the amino acid motifs critical for its binding and antiviral capacities. Therefore, the results presented here provide important information for understanding the entry pathway of SARS-CoV-2 and the design of antivirals that target the membrane fusion step.IMPORTANCE The COVID-19 pandemic, caused by SARS-CoV-2, presents a serious global public health emergency in urgent need of prophylactic and therapeutic interventions. The S protein of coronaviruses mediates viral receptor binding and membrane fusion, thus being considered a critical target for antivirals. Herein, we report that the SARS-CoV-2 S protein has evolved a high level of activity to mediate cell-cell fusion, significantly differing from the S protein of SARS-CoV that emerged previously. The HR1 sequence in the fusion protein of SARS-CoV-2 adopts a much higher helical stability than the HR1 sequence in the fusion protein of SARS-CoV and can interact with the HR2 site to form a six-helical bundle structure more efficiently, underlying the mechanism of the enhanced fusion capacity. Also, importantly, the design of membrane fusion inhibitors with high potencies against both SARS-CoV-2 and SARS-CoV has provided potential arsenals to combat the pandemic and tools to exploit the fusion mechanism.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Lipopeptídeos/farmacologia , Fusão de Membrana/efeitos dos fármacos , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Sequência de Aminoácidos , Betacoronavirus/fisiologia , Desenho de Fármacos , Células HEK293 , Humanos , Lipopeptídeos/química , Glicoproteínas de Membrana/metabolismo , Pandemias , Peptidil Dipeptidase A/metabolismo , Ligação Proteica , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Glicoproteína da Espícula de Coronavírus/metabolismo , Proteínas do Envelope Viral/metabolismo
8.
J Med Chem ; 63(11): 6090-6095, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32378891

RESUMO

A novel lipopeptide antibiotic, stalobacin I (1), was discovered from a culture broth of an unidentified Gram-negative bacterium. Stalobacin I (1) had a unique chemical architecture composed of an upper and a lower half peptide sequence, which were linked via a hemiaminal methylene moiety. The sequence of 1 contained an unusual amino acid, carnosadine, 3,4-dihydroxyariginine, 3-hydroxyisoleucine, and 3-hydroxyaspartic acid, and a novel cyclopropyl fatty acid. The antibacterial activity of 1 against a broad range of drug-resistant Gram-positive bacteria was much stronger than those of "last resort" antibiotics such as vancomycin, linezolid, and telavancin (MIC 0.004-0.016 µg/mL). Furthermore, compound 1 induced a characteristic morphological change in Gram-positive and Gram-negative strains by inflating the bacterial cell body. The absolute configuration of a cyclopropyl amino acid, carnosadine, was determined by the synthetic study of its stereoisomers, which was an essential component for the strong activity of 1.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Lipopeptídeos/química , Aminoglicosídeos/farmacologia , Antibacterianos/química , Avaliação Pré-Clínica de Medicamentos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Lipoglicopeptídeos/farmacologia , Lipopeptídeos/farmacologia , Testes de Sensibilidade Microbiana
9.
Sci Rep ; 10(1): 7269, 2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32350300

RESUMO

Lipidised analgesic peptide prodrugs self-assemble into peptide nanofibers; with the nanofiber morphology protecting the peptide from plasma degradation and improving therapeutic efficacy. Extending this learning, we hypothesised that a self-assembling lipidized peptide arginine vasopressin (AVP) receptor agonist, that had not been designed as a prodrug, could prove pharmacologically active and control urine production. The only approved AVP receptor agonist, desmopressin is indicated for the treatment of central diabetes insipidus (DI), bedwetting, haemophilia A and von Willebrand disease. Desmopressin is well tolerated by most patients, however adverse effects, such as hyponatraemia and water intoxication necessitate a strict fluid intake, thus motivating the search for alternative DI treatments. Selective V2 receptor agonism is required for anti-DI activity and we hypothesised that our new lipidized peptide (METx) would lead to selective AVP receptor agonism. METx was synthesised and characterised and then tested for activity against the V2, V1a and OT uterine receptors and not tested against the V1b receptor as METx was not expected to cross the blood brain barrier. METx was also tested in vivo in a healthy rat model. METx forms nanofibers and is a partial V2 receptor agonist (determined by measuring MDCK cell line cAMP accumulation), producing 57% of AVP's maximal activity (EC50 = 2.7 nM) and is not a V1a agonist up to a concentration of 1 µM (determined by measuring A7r5 cell line D-myo-inositol-1-phosphate accumulation). METx is a weak OT receptor antagonist, reducing the frequency of OT induced contractions (EC50 = 350 nM) and increasing the OT EC50 from 0.081 nM to 21 nM at a concentration of 600 nM. METx (41 nM) had no effect on spontaneous uterine contractions and METx (100 nM) had no effect on OT induced uterine contractions. Simulated binding studies show that binding avidity to the receptors follows the trend: V2 > OT > V1a. On intravenous injection, a nanoparticle formulation of METx reduced urine production in a healthy rat model in a dose responsive manner, with 40 mg kg-1 METx resulting in no urine production over 4 hours. The lipidized self-assembling peptide - METx - is a selective competitive V2 receptor agonist and an anti-diuretic.


Assuntos
Antidiuréticos , Arginina Vasopressina , Lipopeptídeos , Receptores de Vasopressinas/agonistas , Urina , Animais , Antidiuréticos/síntese química , Antidiuréticos/química , Antidiuréticos/farmacologia , Arginina Vasopressina/síntese química , Arginina Vasopressina/química , Arginina Vasopressina/farmacologia , Cães , Feminino , Lipopeptídeos/síntese química , Lipopeptídeos/química , Lipopeptídeos/farmacologia , Células Madin Darby de Rim Canino , Masculino , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Receptores de Vasopressinas/metabolismo
10.
J Virol ; 94(15)2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32404526

RESUMO

We recently reported a group of lipopeptide-based membrane fusion inhibitors with potent antiviral activities against human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV). In this study, the in vivo therapeutic efficacy of such a lipopeptide, LP-52, was evaluated in rhesus macaques chronically infected with pathogenic SIVmac239. In a pilot study with one monkey, monotherapy with low-dose LP-52 rapidly reduced the plasma viral loads to below the limit of detection and maintained viral suppression during three rounds of structurally interrupted treatment. The therapeutic efficacy of LP-52 was further verified in four infected monkeys; however, three out of the monkeys had viral rebounds under the LP-52 therapy. We next focused on characterizing SIV mutants responsible for the in vivo resistance. Sequence analyses revealed that a V562A or V562M mutation in the N-terminal heptad repeat (NHR) and a E657G mutation in the C-terminal heptad repeat (CHR) of SIV gp41 conferred high resistance to LP-52 and cross-resistance to the peptide drug T20 and two newly designed lipopeptides (LP-80 and LP-83). Moreover, we showed that the resistance mutations greatly reduced the stability of diverse fusion inhibitors with the NHR site, and V562A or V562M in combination with E657G could significantly impair the functionality of viral envelopes (Envs) to mediate SIVmac239 infection and decrease the thermostability of viral six-helical bundle (6-HB) core structure. In conclusion, the present data have not only facilitated the development of novel anti-HIV drugs that target the membrane fusion step, but also help our understanding of the mechanism of viral evolution to develop drug resistance.IMPORTANCE The anti-HIV peptide drug T20 (enfuvirtide) is the only membrane fusion inhibitor available for treatment of viral infection; however, it exhibits relatively weak antiviral activity, short half-life, and a low genetic barrier to inducing drug resistance. Design of lipopeptide-based fusion inhibitors with extremely potent and broad antiviral activities against divergent HIV-1, HIV-2, and SIV isolates have provided drug candidates for clinical development. Here, we have verified a high therapeutic efficacy for the lipopeptide LP-52 in SIVmac239-infected rhesus monkeys. The resistance mutations selected in vivo have also been characterized, providing insights into the mechanism of action of newly designed fusion inhibitors with a membrane-anchoring property. For the first time, the data show that HIV-1 and SIV can share a similar genetic pathway to develop resistance, and that a lipopeptide fusion inhibitor could have a same resistance profile as its template peptide.


Assuntos
Lipopeptídeos/farmacologia , Lipoproteínas/farmacologia , Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência Símia/metabolismo , Proteínas Virais de Fusão/metabolismo , Internalização do Vírus/efeitos dos fármacos , Substituição de Aminoácidos , Animais , Lipopeptídeos/química , Lipoproteínas/química , Macaca mulatta , Mutação de Sentido Incorreto , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Síndrome de Imunodeficiência Adquirida dos Símios/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/patologia , Vírus da Imunodeficiência Símia/genética , Proteínas Virais de Fusão/genética
11.
Cell Res ; 30(4): 343-355, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32231345

RESUMO

The recent outbreak of coronavirus disease (COVID-19) caused by SARS-CoV-2 infection in Wuhan, China has posed a serious threat to global public health. To develop specific anti-coronavirus therapeutics and prophylactics, the molecular mechanism that underlies viral infection must first be defined. Therefore, we herein established a SARS-CoV-2 spike (S) protein-mediated cell-cell fusion assay and found that SARS-CoV-2 showed a superior plasma membrane fusion capacity compared to that of SARS-CoV. We solved the X-ray crystal structure of six-helical bundle (6-HB) core of the HR1 and HR2 domains in the SARS-CoV-2 S protein S2 subunit, revealing that several mutated amino acid residues in the HR1 domain may be associated with enhanced interactions with the HR2 domain. We previously developed a pan-coronavirus fusion inhibitor, EK1, which targeted the HR1 domain and could inhibit infection by divergent human coronaviruses tested, including SARS-CoV and MERS-CoV. Here we generated a series of lipopeptides derived from EK1 and found that EK1C4 was the most potent fusion inhibitor against SARS-CoV-2 S protein-mediated membrane fusion and pseudovirus infection with IC50s of 1.3 and 15.8 nM, about 241- and 149-fold more potent than the original EK1 peptide, respectively. EK1C4 was also highly effective against membrane fusion and infection of other human coronavirus pseudoviruses tested, including SARS-CoV and MERS-CoV, as well as SARSr-CoVs, and potently inhibited the replication of 5 live human coronaviruses examined, including SARS-CoV-2. Intranasal application of EK1C4 before or after challenge with HCoV-OC43 protected mice from infection, suggesting that EK1C4 could be used for prevention and treatment of infection by the currently circulating SARS-CoV-2 and other emerging SARSr-CoVs.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/prevenção & controle , Lipopeptídeos/farmacologia , Fusão de Membrana , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Administração Intranasal , Sequência de Aminoácidos , Animais , Betacoronavirus/efeitos dos fármacos , Fusão Celular , Chlorocebus aethiops , Células HEK293 , Humanos , Camundongos , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Vírus da SARS , Alinhamento de Sequência , Relação Estrutura-Atividade , Células Vero
12.
Biofouling ; 36(2): 210-221, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32292058

RESUMO

Surfactin is a type of cyclic lipopeptide biosurfactant implicated in a wide range of applications. Although its antimicrobial activity has been characterized, its effect on Candida albicans physiology remains to be elucidated. The present study evaluated the influence of surfactin-C15 (SF) and its complexes with divalent counterions on C. albicans biofilm formation and preformed biofilms. The SF and metal(II)-SF complexes inhibited biofilm formation and reduced the metabolic activity of mature biofilms in a concentration-dependent manner. The same concentrations of the compounds studied dislodged preexisting biofilms grown on polystyrene plates. Moreover, SF and its metal(II) complexes reduced the mRNA expression of hypha-specific genes HWP1, ALS1, ALS3, ECE1 and SAP4 without exhibiting significant growth inhibition. Further research showed that the compounds tested reduced cellular surface hydrophobicity (CSH). These results suggest that SF and metal(II)-SF complexes could be used as anti-biofilm agents against C. albicans hypha-related infections in clinical practice.


Assuntos
Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Complexos de Coordenação/farmacologia , Hifas/efeitos dos fármacos , Lipopeptídeos/farmacologia , Peptídeos Cíclicos/farmacologia , Tensoativos/metabolismo , Aderência Bacteriana/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Candida albicans/crescimento & desenvolvimento , Candida albicans/metabolismo , Proteínas Fúngicas/genética , Proteínas de Fluorescência Verde/genética , Hifas/crescimento & desenvolvimento
13.
Sci Rep ; 10(1): 6078, 2020 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-32269253

RESUMO

Inflammation of the gastrointestinal tract contributes to the development of inflammatory bowel disease (IBD). Human IBD is modeled by administering dextran sulfate sodium (DSS) to mice. In humans and mice, inflammatory M1 macrophages contribute to the progression of IBD whereas immunosuppressive M2 macrophages protect against colitis. The TLR2/1 agonist PAM3CSK4 (PAM3) induces human and murine monocytes to differentiate into immunosuppressive M2 macrophages, suggesting that PAM3 might be of benefit in the prevention/treatment of colitis. PAM3 was therefore administered to mice treated with DSS. As hypothesized, the number of M2 macrophages rose and disease severity decreased. The critical role of M2 macrophages in this process was established by transferring purified M2 macrophages from PAM3 treated control donors into DSS recipients and reducing colitis. These findings suggest that PAM3 may represent a novel approach to the treatment of human IBD.


Assuntos
Diferenciação Celular , Colite Ulcerativa/tratamento farmacológico , Lipopeptídeos/uso terapêutico , Macrófagos Peritoneais/efeitos dos fármacos , Animais , Células Cultivadas , Colite Ulcerativa/etiologia , Feminino , Lipopeptídeos/farmacologia , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Dodecilsulfato de Sódio/toxicidade , Receptor 2 Toll-Like/agonistas
14.
Mol Pharmacol ; 97(5): 324-335, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32173651

RESUMO

Skin serves not only as a protective barrier to microbial entry into the body but also as an immune organ. The outer layer, the epidermis, is composed predominantly of keratinocytes, which can be stimulated to produce proinflammatory mediators. Although some inflammation is useful to defend against infection, excessive or persistent inflammation can lead to the development of inflammatory skin diseases, such as psoriasis, a common skin disorder affecting approximately 2% of the US population. We have previously found that phosphatidylglycerol (PG) derived from soy can inhibit inflammation in a contact irritant ear edema mouse model. Here, we investigated the ability of soy PG to inhibit inflammatory mediator expression in response to activators of the pattern recognition receptors, toll-like receptor-2 (TLR2) and -4 (TLR4). We found that in epidermal keratinocytes, soy PG inhibited TLR2 and TLR4 activation and inflammatory mediator expression in response to a synthetic triacylated lipopeptide and lipopolysaccharide, respectively, as well as an endogenous danger-associated molecular pattern. However, at higher concentrations, soy PG alone enhanced the expression of some proinflammatory cytokines, suggesting a narrow therapeutic window for this lipid. Dioleoylphosphatidylglycerol (DOPG), but not dioleoylphosphatidylcholine, exerted a similar inhibitory effect, completely blocking keratinocyte inflammatory mediator expression induced by TLR2 and TLR4 activators as well as NFκB activation in a macrophage cell line (RAW264.7); however, DOPG was not itself proinflammatory even at high concentrations. Furthermore, DOPG had no effect on NFκB activation in response to a TLR7/8 agonist. Our results suggest that DOPG could be used to inhibit excessive skin inflammation. SIGNIFICANCE STATEMENT: Although inflammation is beneficial for clearing an infection, in some cases, the infection can be excessive and/or become chronic, thereby resulting in considerable tissue damage and pathological conditions. We show here that the phospholipid phosphatidylglycerol can inhibit the activation of toll-like receptors 2 and 4 of the innate immune system as well as the downstream inflammatory mediator expression in response to microbial component-mimicking agents in epidermal keratinocytes that form the physical barrier of the skin.


Assuntos
Mediadores da Inflamação/metabolismo , Queratinócitos/metabolismo , Padrões Moleculares Associados a Patógenos/farmacologia , Fosfatidilgliceróis/farmacologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Calgranulina B/farmacologia , Humanos , Imidazóis/farmacologia , Lipopeptídeos/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , NF-kappa B/metabolismo , Células RAW 264.7 , Receptores de Reconhecimento de Padrão/metabolismo , Proteínas Recombinantes/farmacologia , Soja/química
15.
Braz Oral Res ; 34: e012, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32049112

RESUMO

Lipoproteins are important bacterial immunostimulating molecules capable of inducing receptor activator of nuclear factor-κB (RANKL) and osteoclast formation in vitro and in vivo . Although these molecules are present in periodontopathogenic bacteria, their role in periodontitis is not known. In this study, we used Pam2CSK4 (PAM2), a synthetic molecule that mimics bacterial lipoprotein, to investigate the effects of lipoproteins on periodontitis in mice. C57BL/6 male mice were randomly divided into three experimental groups: 1) Negative control group: animals received vehicle injection; 2) Positive control group: animals received injection of Escherichia coli lipopolysaccharide (LPS); 3) PAM2 group: animals received PAM2 injection. All the injections were performed bilaterally every other day into the palatal mucosa between first and second molars. After twenty-four days, the animals were euthanized to assess alveolar bone volume (micro-CT), cellular and extracellular composition in the gingiva (stereometric analysis), and osteoclast numbers (TRAP staining). Treatment with either PAM2 or LPS induced gingival inflammation, as demonstrated by increased infiltration of inflammatory cells and enhanced angiogenesis, associated with a smaller number of fibroblasts and decreased extracellular matrix. Importantly, treatment not only with LPS but also with PAM2 resulted in a larger number of TRAP+ multinucleated osteoclasts and significant loss of alveolar bone. Collectively, our data demonstrate that PAM2 can induce gingival inflammation and bone loss in mice, broadening the avenues of investigation into the role of lipoproteins in the pathogenesis of periodontal disease.


Assuntos
Lipopeptídeos/farmacologia , Periodontite/etiologia , Periodontite/patologia , Receptor 2 Toll-Like/antagonistas & inibidores , Perda do Osso Alveolar/etiologia , Perda do Osso Alveolar/patologia , Processo Alveolar/efeitos dos fármacos , Processo Alveolar/patologia , Animais , Modelos Animais de Doenças , Gengiva/efeitos dos fármacos , Gengiva/patologia , Gengivite/etiologia , Gengivite/patologia , Masculino , Camundongos Endogâmicos C57BL , Osteoclastos/efeitos dos fármacos , Osteoclastos/fisiologia , Periodontite/microbiologia , Distribuição Aleatória , Fosfatase Ácida Resistente a Tartarato , Fatores de Tempo , Microtomografia por Raio-X
16.
Mol Immunol ; 120: 52-60, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32065987

RESUMO

Avian infectious bronchitis (IB) is an acute, highly infectious and contagious viral disease of chickens caused by avian infectious bronchitis virus (IBV) belonging to the genus Coronavirus and family Coronaviridae. It can affect all age groups of birds. The toll-like receptors (TLRs) are a major class of innate immune pattern recognition receptors that have a key role in immune response and defense against various infections.The TLRs are essential for initiation of innate immune responses and in the development of adaptive immune responses. An in ovo model was employed to study the antiviral activity of TLR ligands (Pam3CSK4, LPS and CpG ODN) on replication of IBV. It was hypothesized that optimum dose and specific timing of TLR ligands may reduce viral load of IBV in specific pathogen free (SPF) embryonated chicken eggs (ECEs). Further, the mechanism involved in the TLR-mediated antiviral response in chorioallantoic membrane (CAM) of ECEs was investigated. The ECEs of 9-11 days old were treated with different doses (high, intermediate and low) of TLR-2 (Pam3CSK4), TLR-4 (LPS) and TLR-21 (CpG ODN) ligands. In addition, to know the timing of TLR ligand treatment, six time intervals were analyzed viz. 36, 24 and 12 h prior to infection, time of infection (co-administration of TLR ligands and avian IBV) and 12 and 24 h post-IBV infection. For studying the relative expression of immuno-stimulatory genes (IFN-α, IFN-ß, IFN-γ, IL-1ß, iNOS and OAS) in CAM, TLR ligands were administered through intra-allantoicroute and CAM were collected at 4, 8 and 16 h post treatment. The results demonstrated that intermediate dose of all the three TLR ligands significantly reduced virus titers and used in the present study. However, the LPS reduced virus titer pre- and post-IBV infection but Pam3CSK4 and CpG ODN reduced only pre-IBV infection. Further analysis showed that TLR ligands induced IFN-γ, IL-1ß and IFN stimulated genes viz. iNOS and OAS genes in CAM. The present study pointed towards the novel opportunities for rational design of LPS as immuno-stimulatory agent in chickens with reference to IBV. It may be speculated that in ovo administration of these TLR ligands may enhance resistance against viral infection in neonatal chicken and may contribute towards the development of more effective and safer vaccines including in ovo vaccines.


Assuntos
Vírus da Bronquite Infecciosa/imunologia , Receptores Toll-Like/agonistas , Adjuvantes Imunológicos/farmacologia , Animais , Antivirais/farmacologia , Proteínas Aviárias/agonistas , Proteínas Aviárias/imunologia , Embrião de Galinha , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Citocinas/metabolismo , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/imunologia , Imunidade Inata , Vírus da Bronquite Infecciosa/patogenicidade , Vírus da Bronquite Infecciosa/fisiologia , Ligantes , Lipopeptídeos/farmacologia , Lipopolissacarídeos/farmacologia , Oligodesoxirribonucleotídeos/farmacologia , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/prevenção & controle , Doenças das Aves Domésticas/virologia , Receptores Toll-Like/imunologia , Carga Viral/efeitos dos fármacos , Carga Viral/imunologia , Replicação Viral/efeitos dos fármacos , Replicação Viral/imunologia
17.
Planta ; 251(3): 70, 2020 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-32086615

RESUMO

MAIN CONCLUSION: Lipopeptides could help to overcome a large concern in agriculture: resistance against chemical pesticides. These molecules have activity against various phytopathogens and a potential to be transformed by genetic engineering. The exponential rise of pest resistances to different chemical pesticides and the global appeal of consumers for a sustainable agriculture and healthy nutrition have led to the search of new solutions for pest control. Furthermore, new laws require a different stance of producers. Based on that, bacteria of the genus Bacillus present a great agricultural potential, producing lipopeptides (LPs) that have high activity against insects, mites, nematodes, and/or phytopathogens that are harmful to plant cultures. Biopesticide activity can be found mainly in three families of Bacillus lipopeptides: surfactin, iturin, and fengycin. These molecules have an amphiphilic nature, interfering with biological membrane structures. Their antimicrobial properties include activity against bacteria, fungi, oomycetes, and viruses. Recent studies also highlight the ability of these compounds to stimulate defense mechanisms of plants and biofilm formation, which is a key factor for the successful colonization of biocontrol organisms. The use of molecular biology has also recently been researched for continuous advances and discoveries of new LPs, avoiding possible future problems of resistance against these molecules. As a consequence of the properties and possibilities of LPs, numerous studies and developments as well as the attention of large companies in the field is expected in the near future.


Assuntos
Agricultura , Bacillus/metabolismo , Lipopeptídeos/farmacologia , Controle Biológico de Vetores/métodos , Animais , Anti-Infecciosos/farmacologia , Antifúngicos/farmacologia , Bactérias/efeitos dos fármacos , Resistência a Medicamentos , Fungos/efeitos dos fármacos , Insetos/efeitos dos fármacos , Lipopeptídeos/química , Lipopeptídeos/metabolismo , Ácaros/efeitos dos fármacos , Nematoides/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Praguicidas/farmacologia , Doenças das Plantas/prevenção & controle , Plantas/microbiologia , Vírus/efeitos dos fármacos
18.
Molecules ; 25(4)2020 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-32069902

RESUMO

Antimicrobial resistance to conventional antibiotics and the limited alternatives to combat plant-threatening pathogens are worldwide problems. Antibiotic lipopeptides exert remarkable membrane activity, which usually is not prone to fast resistance formation, and often show organism-type selectivity. Additional modes of action commonly complement the bioactivity profiles of such compounds. The present work describes a multicomponent-based methodology for the synthesis of cyclic polycationic lipopeptides with stabilized helical structures. The protocol comprises an on solid support Ugi-4-component macrocyclization in the presence of a lipidic isocyanide. Circular dichroism was employed to study the influence of both macrocyclization and lipidation on the amphiphilic helical structure in water and micellar media. First bioactivity studies against model phytopathogens demonstrated a positive effect of the lipidation on the antimicrobial activity.


Assuntos
Antifúngicos/química , Lactamas/química , Lipopeptídeos/química , Peptídeos Cíclicos/química , Antibacterianos/síntese química , Antibacterianos/química , Antibacterianos/farmacologia , Antifúngicos/síntese química , Antifúngicos/farmacologia , Botrytis/efeitos dos fármacos , Lipopeptídeos/síntese química , Lipopeptídeos/farmacologia , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/farmacologia , Phytophthora infestans/efeitos dos fármacos
19.
Molecules ; 25(2)2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31936341

RESUMO

Ultrashort cationic lipopeptides (USCLs) are promising antimicrobial agents that hypothetically may be alternatively used to combat pathogens such as bacteria and fungi. In general, USCLs consist of fatty acid chains and a few basic amino acid residues. The main shortcoming of USCLs is their relatively high cytotoxicity and hemolytic activity. This study focuses on the impact of the hydrophobic fatty acid chain, on both antimicrobial and hemolytic activities. To learn more about this region, a series of USCLs with different straight-chain fatty acids (C8, C10, C12, C14) attached to the tripeptide with two arginine residues were synthesized. The amino acid at the N-terminal position was exchanged for proteinogenic and non-proteinogenic amino acid residues (24 in total). Moreover, the branched fatty acid residues were conjugated to N-terminus of a dipeptide with two arginine residues. All USCLs had C-terminal amides. USCLs were tested against reference bacterial strains (including ESKAPE group) and Candida albicans. The hemolytic potential was tested on human erythrocytes. Hydrophobicity of the compounds was evaluated by RP-HPLC. Shortening of the fatty acid chain and simultaneous addition of amino acid residue at N-terminus were expected to result in more selective and active compounds than those of the reference lipopeptides with similar lipophilicity. Hypothetically, this approach would also be beneficial to other antimicrobial peptides where N-lipidation strategy was used to improve their biological characteristics.


Assuntos
Aminoácidos/farmacologia , Antibacterianos/farmacologia , Ácidos Graxos/farmacologia , Hemólise/efeitos dos fármacos , Lipopeptídeos/farmacologia , Cátions , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lipopeptídeos/química , Testes de Sensibilidade Microbiana , Relação Estrutura-Atividade
20.
Biochem Biophys Res Commun ; 524(1): 156-162, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-31982133

RESUMO

Formyl peptide receptors (FPRs) are G protein-coupled receptors mainly expressed in inflammatory myeloid cells. Previous reports demonstrated that human neutrophils express only FPR1 and FPR2 but not FPR3. Here, we found that FPR3 is expressed in sepsis patient derived neutrophils and Fpr3 is expressed in the mouse neutrophils. To test the role of Fpr3 in neutrophil activity, we synthesized Fpr3 pepducins and successfully developed an agonistic pepducin that stimulates Fpr3, eliciting calcium increase and chemotactic migration of neutrophils. We also found that administration of an Fpr3 pepducin in an experimental mouse sepsis model significantly increased the survival rate. The pepducin markedly inhibited lung injury, splenocyte apoptosis, and inflammatory cytokine production. Bacterial counts were significantly decreased by the pepducin in septic mice. Based on these results, we suggest that FPR3 can be regarded as a new target to control sepsis, and the newly generated Fpr3-based pepducin can be used for the development of anti-septic agents.


Assuntos
Membrana Celular/metabolismo , Lipopeptídeos/uso terapêutico , Receptores de Formil Peptídeo/metabolismo , Sepse/tratamento farmacológico , Animais , Ceco/patologia , Membrana Celular/efeitos dos fármacos , Citocinas/biossíntese , Células HEK293 , Humanos , Mediadores da Inflamação/metabolismo , Ligadura , Lipopeptídeos/administração & dosagem , Lipopeptídeos/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Punções , Sepse/patologia
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