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1.
J Enzyme Inhib Med Chem ; 38(1): 24-35, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36305272

RESUMO

Ligand-based drug design methods are thought to require large experimental datasets to become useful for virtual screening. In this work, we propose a computational strategy to design novel inhibitors of coronavirus main protease, Mpro. The pipeline integrates publicly available screening and binding affinity data in a two-stage machine-learning model using the recent MACAW embeddings. Once trained, the model can be deployed to rapidly screen large libraries of molecules in silico. Several hundred thousand compounds were virtually screened and 10 of them were selected for experimental testing. From these 10 compounds, 8 showed a clear inhibitory effect on recombinant Mpro, with half-maximal inhibitory concentration values (IC50) in the range 0.18-18.82 µM. Cellular assays were also conducted to evaluate cytotoxic, haemolytic, and antiviral properties. A promising lead compound against coronavirus Mpro was identified with dose-dependent inhibition of virus infectivity and minimal toxicity on human MRC-5 cells.


Assuntos
COVID-19 , Proteases 3C de Coronavírus , Humanos , SARS-CoV-2 , Inibidores de Protease de Coronavírus , Ligantes , Inibidores de Proteases/farmacologia , Inibidores de Proteases/química , Proteínas não Estruturais Virais/metabolismo , Cisteína Endopeptidases/metabolismo , Antivirais/farmacologia , Antivirais/química , Simulação de Acoplamento Molecular
2.
Talanta ; 252: 123824, 2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36027618

RESUMO

Mpro represents one of the most promising drug targets for SARS-Cov-2, as it plays a crucial role in the maturation of viral polyproteins into functional proteins. HTS methods are currently used to screen Mpro inhibitors, and rely on searching chemical databases and compound libraries, meaning that they only consider previously structurally clarified and isolated molecules. A great advancement in the hit identification strategy would be to set-up an approach aimed at exploring un-deconvoluted mixtures of compounds such as plant extracts. Hence, the aim of the present study is to set-up an analytical platform able to fish-out bioactive molecules from complex natural matrices even where there is no knowledge on the constituents. The proposed approach begins with a metabolomic step aimed at annotating the MW of the matrix constituents. A further metabolomic step is based on identifying those natural electrophilic compounds able to form a Michael adduct with thiols, a peculiar chemical feature of many Mpro inhibitors that covalently bind the catalytic Cys145 in the active site, thus stabilizing the complex. A final step consists of incubating recombinant Mpro with natural extracts and identifying compounds adducted to the residues within the Mpro active site by bottom-up proteomic analysis (nano-LC-HRMS). Data analysis is based on two complementary strategies: (i) a targeted search applied by setting the adducted moieties identified as Michael acceptors of Cys as variable modifications; (ii) an untargeted approach aimed at identifying the whole range of adducted peptides containing Cys145 on the basis of the characteristic b and y fragment ions independent of the adduct. The method was set-up and then successfully tested to fish-out bioactive compounds from the crude extract of Scutellaria baicalensis, a Chinese plant containing the catechol-like flavonoid baicalin and its corresponding aglycone baicalein which are well-established inhibitors of Mpro. Molecular dynamics (MD) simulations were carried out in order to explore the binding mode of baicalin and baicalein, within the SARS-CoV-2 Mpro active site, allowing a better understanding of the role of the nucleophilic residues (i.e. His41, Cys145, His163 and His164) in the protein-ligand recognition process.


Assuntos
COVID-19 , SARS-CoV-2 , Animais , Proteases 3C de Coronavírus , Peptídeo Hidrolases , Proteômica , COVID-19/tratamento farmacológico , Inibidores de Proteases/farmacologia , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Simulação de Acoplamento Molecular , Misturas Complexas , Antivirais/farmacologia , Antivirais/química
3.
J Pharm Biomed Anal ; 223: 115118, 2023 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-36332330

RESUMO

Coronavirus disease (COVID-19) caused by SARS-COV-2 infection has been widely prevalent in many countries and has become a common challenge facing mankind. Traditional Chinese medicine (TCM) has played a prominent role in this pandemic, and especially TCM with the function of "heat-clearing and detoxifying" has shown an excellent role in anti-virus. Fufang Shuanghua oral liquid (FFSH) has been used to treat the corresponding symptoms of influenza such as fever, nasal congestion, runny nose, sore throat, and upper respiratory tract infections in clinic, which are typical symptoms of COVID-19. The content of chlorogenic acid, andrographolide and dehydrated andrographolide as the quality control components of FFSH is not less than 1.0 mg/mL, 60 µg/mL and 60 µg/mL respectively. In this study, UPLC-Q-TOF-MS/MS was employed to describe the chemical profile of FFSH. Virtual screening and fluorescence resonance energy transfer (FRET) were used to screen the effective components of FFSH acting on SARS-CoV-2 main protease (Mpro). As a result, 214 compounds in FFSH were identified or preliminarily characterized by UPLC-Q-TOF-MS/MS, and 61 active ingredients with potential inhibitory effects on Mpro were selected through receptor-based and ligand-based virtual screening. In particular, quercetin, forsythoside A, and linoleic acid showed a good inhibitory effect on Mpro in FRET evaluation with IC50 values of 26.15 µM, 22.26 µM and 47.09 µM respectively, and had a strong binding affinity with the receptor Mpro (6LU7) in molecular docking. CYS145 and HIS41 were the main amino acid residues affected by small molecules in the protein binding domain. In brief, we characterized, for the first time, 214 chemical components in FFSH, and three of them, including quercetin, forsythoside A and linoleic acid, were screened out to exert beneficial anti-COVID-19 effects through CYS145 and HIS41 sites, which may provide a new research strategy for TCM to develop new therapeutic drugs against COVID-19.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , COVID-19/tratamento farmacológico , Simulação de Acoplamento Molecular , Peptídeo Hidrolases , Quercetina/farmacologia , Espectrometria de Massas em Tandem , Ácido Linoleico , Proteínas não Estruturais Virais , Inibidores de Proteases/farmacologia
4.
J Ethnopharmacol ; 300: 115694, 2023 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-36096346

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Libidibia ferrea (Mart. ex. Tul.) L.P. Queiroz is a Brazilian native tree locally known as jucá and pau-ferro, and it has been used in folk medicine for relieving, asthma, bronchitis, sore throat, rheumatism, enterocolitis and fever. The anti-inflammatory properties of L. ferrea were confirmed for its stem, fruit, leaves, bark and seeds extracts, however little is known about the natural compounds that may be associated with that response. AIM OF THIS STUDY: In a normal physiological condition, many enzymes play an important role in catalyzing biological functions. Among them, proteases are of great interest. Although they take part of many biological systems, as the inflammatory process, when deregulated, proteases may cause system malfunctions, such as under- or overproduction of cytokines, or immune cells activation. Thus, protease inhibitors prevent these immune responses by regulating proteases. The objective of this study was to evaluate the anti-inflammatory and anti-nociceptive response of a protease inhibitor purified from L. ferrea seeds (LfTI). MATERIALS AND METHODS: In vitro (5, 50 and 250 µg/mL of LfTI) and in vivo (0.6, 3 e 15 mg/kg of LfTI) assays were performed. Male Swiss mice weighing 18-25 g were used for cell harvesting and for the in vivo assays. The anti-inflammatory activity was analyzed in vitro by macrophage cytotoxicity, hydrogen peroxide (H2O2) production, and cell adhesion assays; and in vivo by leukocyte recruitment, nitric oxide (NO) production, vascular permeability, paw edema and mast cell degranulation assays. The anti-nociceptive activity was evaluated through abdominal writhing test induced by acetic acid and formalin sensitization. RESULTS: Our results showed that, in vitro, LfTI is not cytotoxic. Also, LfTI (50 µg/mL) inhibited macrophage H2O2 production (48.2%), and adhesion (48.4%). LfTI (0.6, 3 e 15 mg/kg) decreased polymorphonuclear cell recruitment dose-dependently, and it inhibited NO production (53%), vascular permeability (40.7%) and paw edema at 3 mg/kg at different time, but it did not inhibit mast cell degranulation. Besides, LfTI did not inhibit either the number of writhing or the licking time in the formalin test in the second phase (inflammatory). However, LfTI (3 mg/kg) inhibited licking time at the first phase (neurogenic) in the formalin sensitization (46.1%). CONCLUSIONS: Our results show that LfTI has anti-inflammatory and antinociceptive (neurogenic pain) effects, and these effects might be associated with the inhibition of inflammatory proteases and/or protease-activated receptors activation hindering.


Assuntos
Anti-Infecciosos , Caesalpinia , Analgésicos/efeitos adversos , Animais , Anti-Infecciosos/uso terapêutico , Anti-Inflamatórios/efeitos adversos , Citocinas , Edema/tratamento farmacológico , Formaldeído , Peróxido de Hidrogênio , Camundongos , Óxido Nítrico , Peptídeo Hidrolases , Extratos Vegetais/efeitos adversos , Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico , Receptores Ativados por Proteinase/uso terapêutico , Sementes
5.
J Am Chem Soc ; 144(46): 21035-21045, 2022 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-36356199

RESUMO

Given the current impact of SARS-CoV2 and COVID-19 on human health and the global economy, the development of direct acting antivirals is of paramount importance. Main protease (MPro), a cysteine protease that cleaves the viral polyprotein, is essential for viral replication. Therefore, MPro is a novel therapeutic target. We identified two novel MPro inhibitors, D-FFRCMKyne and D-FFCitCMKyne, that covalently modify the active site cysteine (C145) and determined cocrystal structures. Medicinal chemistry efforts led to SM141 and SM142, which adopt a unique binding mode within the MPro active site. Notably, these inhibitors do not inhibit the other cysteine protease, papain-like protease (PLPro), involved in the life cycle of SARS-CoV2. SM141 and SM142 block SARS-CoV2 replication in hACE2 expressing A549 cells with IC50 values of 8.2 and 14.7 nM. Detailed studies indicate that these compounds also inhibit cathepsin L (CatL), which cleaves the viral S protein to promote viral entry into host cells. Detailed biochemical, proteomic, and knockdown studies indicate that the antiviral activity of SM141 and SM142 results from the dual inhibition of MPro and CatL. Notably, intranasal and intraperitoneal administration of SM141 and SM142 lead to reduced viral replication, viral loads in the lung, and enhanced survival in SARS-CoV2 infected K18-ACE2 transgenic mice. In total, these data indicate that SM141 and SM142 represent promising scaffolds on which to develop antiviral drugs against SARS-CoV2.


Assuntos
COVID-19 , Hepatite C Crônica , Animais , Camundongos , Humanos , Antivirais/farmacologia , Antivirais/uso terapêutico , Antivirais/química , Proteases 3C de Coronavírus , Catepsina L/química , Catepsina L/metabolismo , RNA Viral , SARS-CoV-2 , COVID-19/tratamento farmacológico , Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico , Inibidores de Proteases/química , Peptídeo Hidrolases , Proteômica , Proteínas não Estruturais Virais/química , Simulação de Acoplamento Molecular
6.
Antiviral Res ; 208: 105458, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36336176

RESUMO

Severe acute respiratory syndrome coronaviruses 1 and 2 (SARS-CoV-1 and SARS-CoV-2) pose a threat to global public health. The 3C-like main protease (Mpro), which presents structural similarity with the active site domain of enterovirus 3C protease, is one of the best-characterized drug targets of these viruses. Here we studied the antiviral activity of the orally bioavailable enterovirus protease inhibitor AG7404 against SARS-CoV-1 and SARS-CoV-2 from a structural, biochemical, and cellular perspective, comparing it with the related molecule rupintrivir (AG7800). Crystallographic structures of AG7404 in complex with SARS-CoV-1 Mpro and SARS-CoV-2 Mpro and of rupintrivir in complex with SARS-CoV-2 Mpro were solved, revealing that all protein residues interacting with the inhibitors are conserved between the two proteins. A detailed analysis of protein-inhibitor interactions indicates that AG7404 has a better fit to the active site of the target protease than rupintrivir. This observation was further confirmed by biochemical FRET assays showing IC50 values of 47 µM and 101 µM for AG7404 and rupintrivir, respectively, in the case of SARS-CoV-2 Mpro. Equivalent IC50 values for SARS-CoV-1 also revealed greater inhibitory capacity of AG7404, with a value of 29 µM vs. 66 µM for rupintrivir. Finally, the antiviral activity of the two inhibitors against SARS-CoV-2 was confirmed in a human cell culture model of SARS-CoV-2 infection, although rupintrivir showed a higher potency and selectivity index in this assay.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Antivirais/química , COVID-19/tratamento farmacológico , Cisteína Endopeptidases/metabolismo , Inibidores de Proteases/farmacologia , Inibidores de Proteases/química , Simulação de Acoplamento Molecular
7.
Eur J Med Chem ; 244: 114853, 2022 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-36332546

RESUMO

SARS-CoV-2 caused worldwide the current outbreak called COVID-19. Despite multiple countermeasures implemented, there is an urgent global need for new potent and efficient antiviral drugs against this pathogen. In this context, the main protease (Mpro) of SARS-CoV-2 is an essential viral enzyme and plays a pivotal role in viral replication and transcription. Its specific cleavage of polypeptides after a glutamine residue has been considered as a key element to design novel antiviral drugs. Herein, we reported the design, synthesis and structure-activity relationships of novel α-ketoamides as covalent reversible inhibitors of Mpro, exploiting the PADAM oxidation route. The reported compounds showed µM to nM activities in enzymatic and in the antiviral cell-based assays against SARS-CoV-2 Mpro. In order to assess inhibitors' binding mode, two co-crystal structures of SARS-CoV-2 Mpro in complex with our inhibitors were solved, which confirmed the covalent binding of the keto amide moiety to the catalytic Cys145 residue of Mpro. Finally, in order to interrogate potential broad-spectrum properties, we assessed a selection of compounds against MERS Mpro where they showed nM inhibitory potency, thus highlighting their potential as broad-spectrum coronavirus inhibitors.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Proteases 3C de Coronavírus , COVID-19/tratamento farmacológico , Inibidores de Proteases/farmacologia , Inibidores de Proteases/química , Proteínas não Estruturais Virais , Cisteína Endopeptidases/metabolismo , Antivirais/farmacologia , Antivirais/química , Simulação de Acoplamento Molecular
8.
Biochemistry ; 61(22): 2495-2505, 2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36326185

RESUMO

The main protease (Mpro) of SARS-CoV-2 is essential for viral replication and has been the focus of many drug discovery efforts since the start of the COVID-19 pandemic. Nirmatrelvir (NTV) is an inhibitor of SARS-CoV-2 Mpro that is used in the combination drug Paxlovid for the treatment of mild to moderate COVID-19. However, with increased use of NTV across the globe, there is a possibility that future SARS-CoV-2 lineages will evolve resistance to NTV. Early prediction and monitoring of resistance mutations could allow for measures to slow the spread of resistance and for the development of new compounds with activity against resistant strains. In this work, we have used in silico mutational scanning and inhibitor docking of Mpro to identify potential resistance mutations. Subsequent in vitro experiments revealed five mutations (N142L, E166M, Q189E, Q189I, and Q192T) that reduce the potency of NTV and of a previously identified non-covalent cyclic peptide inhibitor of Mpro. The E166M mutation reduced the half-maximal inhibitory concentration (IC50) of NTV 24-fold and 118-fold for the non-covalent peptide inhibitor. Our findings inform the ongoing genomic surveillance of emerging SARS-CoV-2 lineages.


Assuntos
Antivirais , COVID-19 , Proteases 3C de Coronavírus , Farmacorresistência Viral , Inibidores de Proteases , SARS-CoV-2 , Humanos , Antivirais/farmacologia , Antivirais/química , COVID-19/tratamento farmacológico , COVID-19/virologia , Simulação de Acoplamento Molecular , Mutação , Pandemias , Inibidores de Proteases/farmacologia , Inibidores de Proteases/química , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/genética , Farmacorresistência Viral/genética , Proteases 3C de Coronavírus/antagonistas & inibidores
9.
J Chem Phys ; 157(18): 185101, 2022 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-36379777

RESUMO

The main protease (Mpro) of SARS-CoV-2 is an essential enzyme for the replication of the virus causing the COVID-19 pandemic. Because there is no known homologue in humans, it has been proposed as a primary target for antiviral drug development. Here, we explore the potential of five acrylamide-based molecules as possible covalent inhibitors, leading to target MPro by docking, followed by polarizable molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) calculations. All calculations involving a classical potential were calculated with the AMOEBABIO18 polarizable force field, while electronic structure calculations were performed within the framework of density functional theory. Selected docking poses for each of the five compounds were used for MD simulations, which suggest only one of the tested leads remains bound in a catalytically active orientation. The QM/MM results for the covalent attachment of the promising lead to the catalytic serine suggest that this process is thermodynamically feasible but kinetically unlikely. Overall, our results are consistent with the low labeling percentages determined experimentally and may be useful for further development of acrylamide-based leads.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Pandemias , Proteases 3C de Coronavírus , Simulação de Dinâmica Molecular , Peptídeo Hidrolases/metabolismo , Acrilamida , Inibidores de Proteases/farmacologia , Inibidores de Proteases/química , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Simulação de Acoplamento Molecular
10.
Int J Mol Sci ; 23(22)2022 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-36430785

RESUMO

Molecular phenotypes induced by environmental stimuli can be transmitted to offspring through epigenetic inheritance. Using transcriptome profiling, we show that the adaptation of Helicoverpa armigera larvae to soybean peptidase inhibitors (SPIs) is associated with large-scale gene expression changes including the upregulation of genes encoding serine peptidases in the digestive system. Furthermore, approximately 60% of the gene expression changes induced by SPIs persisted in the next generation of larvae fed on SPI-free diets including genes encoding regulatory, oxidoreductase, and protease functions. To investigate the role of epigenetic mechanisms in regulating SPI adaptation, the methylome of the digestive system of first-generation larvae (fed on a diet with and without SPIs) and of the progeny of larvae exposed to SPIs were characterized. A comparative analysis between RNA-seq and Methyl-seq data did not show a direct relationship between differentially methylated and differentially expressed genes, while trypsin and chymotrypsin genes were unmethylated in all treatments. Rather, DNA methylation potential epialleles were associated with transcriptional and translational controls; these may play a regulatory role in the adaptation of H. armigera to SPIs. Altogether, our findings provided insight into the mechanisms of insect adaptation to plant antiherbivore defense proteins and illustrated how large-scale transcriptional reprograming of insect genes can be transmitted across generations.


Assuntos
Mariposas , Soja , Animais , Soja/genética , Soja/metabolismo , Inibidores de Proteases/farmacologia , Regulação para Cima , Serina Proteases/metabolismo , Mariposas/genética , Mariposas/metabolismo , Quimotripsina/genética , Quimotripsina/metabolismo , Tripsina/metabolismo , Larva/genética , Larva/metabolismo , Serina/metabolismo
11.
Antiviral Res ; 208: 105450, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36354082

RESUMO

FB2001 is a drug candidate that targets the main protease of SARS-CoV-2 via covalently binding to cysteine 145. In this study, we evaluated the inhibitory activities of FB2001 against several SARS-CoV-2 variants in vitro and in vivo (in mice), and we also evaluated the histopathological analysis and immunostaining of FB2001 on lung and brain which have been rarely reported. The results showed that FB2001 exhibited potent antiviral efficacy against several current SARS-CoV-2 variants in Vero E6 cells, namely, B.1.1.7 (Alpha): EC50 = 0.39 ± 0.01 µM, EC90 = 0.75 ± 0.01 µM; B.1.351 (Beta): EC50 = 0.28 ± 0.11 µM, EC90 = 0.57 ± 0.21 µM; B.1.617.2 (Delta): EC50 = 0.27 ± 0.05 µM, EC90 = 0.81 ± 0.20 µM; B.1.1.529 (Omicron): EC50 = 0.26 ± 0.06 µM and EC50 = 0.042 ± 0.007 µM (in the presence of a P-glycoprotein inhibitor). FB2001 remained potent against SARS-CoV-2 replication in the presence of high concentrations of human serum, which indicating that human serum had no significant effect on the in vitro inhibitory activity. Additionally, this inhibitor exhibited an additive effect against SARS-CoV-2 when combined with Remdesivir. Furthermore, FB2001 significantly reduced the SARS-CoV-2 copy numbers and titers in the lungs and brains in vivo, and alleviated the pathological symptoms. In addition, FB2001 could alleviated local bleeding, erythrocyte overflow, edema, and inflammatory cell infiltration in brain tissue, and inhibitors reducing viral titers and improving inflammation in the brain have been rarely reported. A physiologically based pharmacokinetic model was established and verified to predict the FB2001 concentration in human lungs. When FB2001 was administered at 200 mg twice a day for 5 days, the observed Ctrough ss in plasma and predicted Ctrough ss of lung total concentration were 0.163 and 2.5 µg/mL, which were approximately 9 and 132-fold higher than the EC50 of 0.019 µg/mL (0.042 µM) against Omicron variant. Taken together, our study suggests that FB2001 is a promising therapeutic agent in COVID-19 treatment and can be combined with remdesivir to achieve improved clinical outcomes. Owing to its good safety and tolerability in healthy human (NCT05197179 and NCT04766931), FB2001 has been approved for Phase II/III clinical trial (NCT05445934).


Assuntos
COVID-19 , Infecções por Coronavirus , Pneumonia Viral , Animais , Humanos , Camundongos , SARS-CoV-2 , Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico , Betacoronavirus , COVID-19/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Infecções por Coronavirus/tratamento farmacológico , Pandemias , Antivirais/farmacologia , Antivirais/uso terapêutico
12.
STAR Protoc ; 3(4): 101794, 2022 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-36317181

RESUMO

Discovery of efficacious antiviral agents targeting SARS-CoV-2 main protease (Mpro) is of the highest importance to fight against COVID-19. Here, we describe a simple protocol for high-throughput screening of Mpro inhibitors using a robust fluorescence polarization (FP) assay. Candidate Mpro inhibitors from large compound libraries could be rapidly identified by monitoring the change of millipolarization unit value. This affordable FP assay can be modified to screen antiviral agents targeting virus protease. For complete details on the use and execution of this protocol, please refer to Li et al. (2022), Yan et al. (2021), and Yan et al. (2022c).


Assuntos
COVID-19 , Ensaios de Triagem em Larga Escala , Humanos , COVID-19/tratamento farmacológico , SARS-CoV-2 , Proteínas não Estruturais Virais , Cisteína Endopeptidases , Inibidores de Proteases/farmacologia , Antivirais/farmacologia , Polarização de Fluorescência
13.
Int J Biol Sci ; 18(16): 6008-6019, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36439874

RESUMO

Cardiac fibrosis is one of the common pathological processes in many cardiovascular diseases characterized by excessive extracellular matrix deposition. SerpinE2 is a kind of protein that inhibits peptidase in extracellular matrix and up-regulated tremendously in mouse model of cardiac fibrosis induced by pressure-overloaded via transverse aortic constriction (TAC) surgery. However, its effect on cardiac fibroblasts (CFs), collagen secretion and the underlying mechanism remains unclear. In this study, DyLight® 488 green fluorescent dye or His-tagged proteins were used to label the exogenous serpinE2 protein. It was showed that extracellular serpinE2 translocated into CFs by low-density lipoprotein receptor-related protein 1 (LRP1) and urokinase plasminogen activator receptor (uPAR) of cell membrane through endocytosis. Knockdown of LRP1 or uPAR reduced the level of serpinE2 in CFs and down-regulated the collagen expression. Inhibition of the endocytosis of serpinE2 could inhibit ERK1/2 and ß-catenin signaling pathways and subsequently attenuated collagen secretion. Knockdown of serpinE2 attenuates cardiac fibrosis in TAC mouse. We conclude that serpinE2 could be translocated into cardiac fibroblasts due to endocytosis through directly interact with the membrane protein LRP1 and uPAR, and this process activated the ERK1/2, ß-catenin signaling pathways, consequently promoting collagen production.


Assuntos
Síndrome de Fadiga Crônica , beta Catenina , Camundongos , Animais , beta Catenina/metabolismo , Serpina E2/metabolismo , Serpina E2/farmacologia , Inibidores de Proteases/farmacologia , Sistema de Sinalização das MAP Quinases/genética , Fibrose , Transdução de Sinais/genética , Endocitose/genética , Colágeno/metabolismo
14.
MEDICC Rev ; 24(3-4): 46-52, 2022 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-36417334

RESUMO

INTRODUCTION: In inflammatory respiratory diseases, the imbalance between proteases and endogenous protease inhibitors leads to an exacerbated activity of human neutrophil elastase (a protease that destroys the extracellular matrix and stimulates proinflammatory cytokine release). Elastase is considered a target in the search for therapeutic treatments for inflammatory respiratory diseases. Pulmonary surfactant is a promising product for this purpose, because in addition to its biophysical function, it has anti-inflammatory properties. OBJECTIVE: Evaluate effect of the Cuban porcine pulmonary surfactant (Surfacen), the rCmPI-II elastase inhibitor, and the Surfacen/rCmPI-II combination on activated neutrophil elastase activity in vitro, and determine if Surfacen's interface property changes in the presence of the inhibitor. METHODS: The anti-elastase effect of Surfacen, rCmPI-II and the Surfacen/rCmPI-II combination was evaluated in an in vitro model of activated neutrophils, previously purified from the blood of healthy subjects. The cells were stimulated with LPS/fMLP and were incubated with different concentrations of Surfacen, rCmPI-II and the Surfacen/rCmPI-II combination. Elastase activity was measured. The interface property was determined on a Langmuir surface balance. The new index, called the abdominal adipose deposit index, was obtained by multiplying the subcutaneous fat thickness by visceral fat thickness, both measured by ultrasound. A cutoff point was established that facilitated discernment of an unhealthy phenotype: normal weight but metabolically obese, a cardiometabolic risk factor. RESULTS: Surfacen at 10 mg/mL inhibited 71% of stimulated neutrophil elastase activity. rCmPI-II at 0.1 µM reduced 20% of elastase activity; at 200 µM-the maximum concentration evaluated-inhibition was 68%. Both products had a dose-dependent effect. The Surfacen/inhibitor combination (0.5 mg/mL/80 µM) did not affect the surfactant interface property or the inhibitory activity of rCmPI-II against human neutrophil elastase. CONCLUSIONS: Surfacen and the rCmPI-II inhibitor have an anti-elastase effect on an activated neutrophil model. rCmPI-II does not affect Surfacen's interface property and, therefore, both can be evaluated for combined use in treating inflammatory lung diseases.


Assuntos
Elastase de Leucócito , Surfactantes Pulmonares , Animais , Humanos , Antivirais , Elastase de Leucócito/farmacologia , Neutrófilos , Inibidores de Proteases/farmacologia , Surfactantes Pulmonares/farmacologia , Suínos
15.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 53(5): 851-856, 2022 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-36224688

RESUMO

Objective: To investigate the distribution characteristics of the HIV genetic subtypes and the status quo of transmitted drug resistance among HIV/AIDS patients in Sichuan with no previous history of receiving antiretroviral therapy (ART). Methods: Adult HIV/AIDS patients who were hospitalized in Sichuan and who had no previous history of exposure to ART drugs exposure were enrolled. In-house sequencing of the HIV gene was done and phylogenetic tree was constructed to analyze the HIV genetic subtypes. The Stanford HIV drug resistance database was used to make online comparison of the drug resistance mutation sites and to determine the presence or absence of drug resistance, and the type and level of drug resistance. Results: A total of 120 patients were enrolled for the study, and 120 blood samples were collected. The genetic subtypes of 87.5% (105/120) of the samples were successfully amplified. The distribution characteristics of HIV genotype were as follows, CRF01_AE accounted for 46.67% (49/105), CRF07_BC accounted for 39.05% (41/105), and the others genetic subtypes, 14.28% (15/105). There were no significant differences between the different genetic subtypes in sex, age, ethnicity, HIV transmission route, drug resistance, baseline HIV RNA and baseline CD4 ( P>0.05). Drug-resistant mutation sites were detected in 25 samples, accounting for 20.83% (25/120) of all samples, with 16.67% (20/120) being potential drug resistance and 4.17% (5/120) being transmitted drug resistance. For the 24 samples found to be resistant to non-nucleoside reverse transcriptase inhibitors (NNRTIs), the mutation frequency of V179D/E was the highest. One patient showed resistance to protease inhibitors (PI) and the mutation site was M46I. No nucleoside reverse transcriptase inhibitor (NRTI) or integrase inhibitors (INTI) resistance were found. Conclusions: The main genetic subtypes of HIV/AIDS patients in Sichuan with no previous history of receiving ART were CRF01_AE and CRF07_BC. The incidence of transmitted drug resistance was low. The drug resistance detected in the study was predominantly resistance to NNRTIs. Baseline HIV drug resistance testing is of great significance for formulating effective ART regimens.


Assuntos
Síndrome de Imunodeficiência Adquirida , Infecções por HIV , HIV-1 , Síndrome de Imunodeficiência Adquirida/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida/epidemiologia , Adulto , China/epidemiologia , Farmacorresistência Viral/genética , Genótipo , Infecções por HIV/tratamento farmacológico , Infecções por HIV/epidemiologia , HIV-1/genética , Humanos , Inibidores de Integrase/farmacologia , Inibidores de Integrase/uso terapêutico , Mutação , Filogenia , Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico , RNA/farmacologia , RNA/uso terapêutico , Inibidores da Transcriptase Reversa/farmacologia , Inibidores da Transcriptase Reversa/uso terapêutico
16.
Pak J Pharm Sci ; 35(4(Special)): 1241-1250, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-36218103

RESUMO

The bacterial HslVU complex consists of two different proteins, i.e., the HslV protease and the HslU ATPase. The functional HslVU enzyme complex forms only when the HslU c-terminal helix is inserted into the cavity located between two adjacent HslV monomers in order to allosterically activate the HslV protease. Based on its essential role in maintaining microbial proteostasis as well its absence from human beings, it is considered a promising therapeutic target for designing antibacterial agents. The goal of the present study was to find out potential drug candidates that could over-activate the HslV protease and produce aberrant proteolysis in pathogenic bacteria. Derivatives of 3-substituted coumarin have been identified as potential HslV protease activators based on their highest docking scores, ideal interaction patterns, and significant in-vitro HslV activation potential. Their ED50 values were in the sub-micromolar range, i.e., 0.4-0.48µM. The conformational stability of the contacts between the HslV dimer and the active compounds was further confirmed by molecular dynamics studies. Correspondingly, the ADMET characteristics of these lead molecules considerably demonstrated their significant non-toxic drug-like abilities. This research not only identified small non-peptidic HslV protease activators but also improved the understanding of the mode of action of 3-substituted coumarin derivatives as antibacterials.


Assuntos
Proteínas de Bactérias , Cumarínicos , Endopeptidases , Peptídeo Hidrolases , Inibidores de Proteases , Adenosina Trifosfatases/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Cumarínicos/farmacologia , Endopeptidases/metabolismo , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/farmacologia
17.
Int J Mol Sci ; 23(20)2022 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-36293408

RESUMO

The tomato yellow leaf curl virus (TYLCV) is the causal agent of one of the most severe diseases affecting tomato growth; however, nitric oxide (NO) can mediate plant resistance. This study investigated the molecular mechanism of exogenous NO donor-mediated disease resistance in tomato seedlings. Tomato seedlings were treated with sodium nitroprusside and TYLCV and subjected to phenotypic, transcriptomic, and physiological analyses. The results show that exogenous NO significantly reduced disease index, MDA content, and virus content (71.4%), significantly increased stem length and fresh weight of diseased plants (p < 0.05), and improved photosynthesis with an induction effect of up to 44.0%. In this study, it was found that the reduction in virus content caused by the increased expression of peptidase inhibitor genes was the main reason for the increased resistance in tomatoes. The peptidase inhibitor inhibited protease activity and restrained virus synthesis, while the significant reduction in virus content inevitably caused a partial weakening or shutdown of the disease response process in the diseased plant. In addition, exogenous NO also induces superoxide dismutase, peroxidase activity, fatty acid elongation, resistance protein, lignin, and monoterpene synthesis to improve resistance. In summary, exogenous NO enhances resistance in tomatoes mainly by regulating peptidase inhibitor genes.


Assuntos
Begomovirus , Lycopersicon esculentum , Óxido Nítrico , Inibidores de Proteases/farmacologia , Nitroprussiato/farmacologia , Lignina , Doenças das Plantas/genética , Begomovirus/genética , Plântula/genética , Superóxido Dismutase , Monoterpenos , Peroxidases , Ácidos Graxos , Peptídeo Hidrolases
18.
J Mol Model ; 28(11): 365, 2022 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-36274116

RESUMO

Dengue fever has been a global health concern. Mitigation is a challenging problem due to non-availability of workable treatments. The most difficult objective is to design a perfect anti-dengue agent capable of inhibiting infections caused by all four serotypes. Various tactics have been employed in the past to discover dengue antivirals, including screening of chemical compounds against dengue virus enzymes. The objective of the current study is to investigate phytocompounds as anti-dengue remedies that target the non-structural 2B and non-structural 3 protease (NS2B-NS3pro), a possible therapeutic target for dengue fever. Initially, 300 + antiviral phytocompounds were collected from Duke's phytochemical and ethnobotanical database and 30 phytocompounds with anti-dengue properties were identified from previously reported studies, which were virtually screened against NS2B-NS3pro using molecular docking and toxicity evaluation. The top five most screened ligands were naringin, hesperidin, gossypol, maslinic acid and rhodiolin with binding affinities of - 8.7 kcal/mol, - 8.5 kcal/mol, - 8.5 kcal/mol, - 8.5 kcal/mol and - 8.1 kcal/mol, respectively. The finest docked compounds complexed with NS2B-NS3pro were subjected for molecular dynamics (MD) simulations and binding free energy estimations through molecular mechanics generalized born surface area-based calculations. The results of the study are intriguing in the context of computer-aided screening and the binding affinities of the phytocompounds, proposing maslinic acid (MAS) as a potent bioactive antiviral for the development of phytocompound-based anti-dengue agent.


Assuntos
Vírus da Dengue , Dengue , Gossipol , Hesperidina , Humanos , Simulação de Acoplamento Molecular , Antivirais/farmacologia , Antivirais/química , Simulação de Dinâmica Molecular , Proteínas não Estruturais Virais/química , Vírus da Dengue/metabolismo , Peptídeo Hidrolases/metabolismo , Compostos Fitoquímicos , Dengue/tratamento farmacológico , Inibidores de Proteases/farmacologia , Inibidores de Proteases/química
19.
Eur J Med Chem ; 244: 114803, 2022 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-36209629

RESUMO

SARS-CoV-2 3CL protease is one of the key targets for drug development against COVID-19. Most known SARS-CoV-2 3CL protease inhibitors act by covalently binding to the active site cysteine. Yet, computational screens against this enzyme were mainly focused on non-covalent inhibitor discovery. Here, we developed a deep learning-based stepwise strategy for selective covalent inhibitor screen. We used a deep learning framework that integrated a directed message passing neural network with a feed-forward neural network to construct two different classifiers for either covalent or non-covalent inhibition activity prediction. These two classifiers were trained on the covalent and non-covalent 3CL protease inhibitors dataset, respectively, which achieved high prediction accuracy. We then successively applied the covalent inhibitor model and the non-covalent inhibitor model to screen a chemical library containing compounds with covalent warheads of cysteine. We experimentally tested the inhibition activity of 32 top-ranking compounds and 12 of them were active, among which 6 showed IC50 values less than 12 µM and the strongest one inhibited SARS-CoV-2 3CL protease with an IC50 of 1.4 µM. Further investigation demonstrated that 5 of the 6 active compounds showed typical covalent inhibition behavior with time-dependent activity. These new covalent inhibitors provide novel scaffolds for developing highly active SARS-CoV-2 3CL covalent inhibitors.


Assuntos
COVID-19 , Aprendizado Profundo , Humanos , SARS-CoV-2 , Inibidores de Proteases/farmacologia , Inibidores de Proteases/química , Proteases 3C de Coronavírus , COVID-19/tratamento farmacológico , Cisteína , Antivirais/farmacologia
20.
Arch Razi Inst ; 77(2): 843-852, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-36284983

RESUMO

Nowadays dengue virus infection (DENV) is one of the major health complications in the world. Although DENV is an old and common disease, unfortunately, until now, there are no specific relevant treatments available for it. This study, therefore, aimed to design, as well as synthesize selective peptide inhibitors, and investigate their activity by in-vitro NS2B/NS3 protease inhibition assay. The design of the peptide ligands was based on studying the interactions with the dengue NS2B/NS3 protease using the computational docking technique in the MOE and AutoDock (version 4.2) software. To this end, the researchers designed 26 linear pentapeptides based on previous studies. It was revealed that two linear pentapeptides (i.e., GKRRK and KRRRK) are the best potential inhibitors. Furthermore, based on the findings of the two independent docking programs, the peptide GKRRK was synthesized by solid-phase peptide synthesis and its structure was confirmed. The in-vitro protease inhibitor study was conducted for these two peptides to examine their activity against the dengue virus using a protin in as a control. It was found that the designed potential peptides possess interesting inhibition against the NS2B/NS3 protease. Additionally, the findings showed that the peptide GKRRK had the highest percentage of inhibition (71.11%) at 100 µM with the IC50 of 48.87 µM; therefore, this linear peptide could serve as a good inhibitor for the DENV.


Assuntos
Vírus da Dengue , Dengue , Animais , Inibidores de Proteases/farmacologia , Inibidores de Proteases/química , Peptídeo Hidrolases , Simulação de Acoplamento Molecular , Peptídeos/farmacologia , Dengue/veterinária
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