BACKGROUND Hepatitis C virus genotype 3a(HCV G3a)is highly prevalent in Pakistan.Due to the elevated cost of available Food and Drug Administration-approved drugs against HCV,medicinal natural products of potent antiv...BACKGROUND Hepatitis C virus genotype 3a(HCV G3a)is highly prevalent in Pakistan.Due to the elevated cost of available Food and Drug Administration-approved drugs against HCV,medicinal natural products of potent antiviral activity should be screened for the cost-effective treatment of the disease.Furthermore,from natural products,active compounds against vital HCV proteins like non-structural protein 3(NS3)protease could be identified to prevent viral proliferation in the host.AIM To develop cost-effective HCV genotype 3a NS3 protease inhibitors from citrus fruit extracts.METHODS Full-length NS3 without co-factor non-structural protein 4A(NS4A)and codon optimized NS3 protease in fusion with NS4A were expressed in Escherichia coli.The expressed protein was purified by metal ion affinity chromatography and gel filtration.Citrus fruit extracts were screened using fluorescence resonance energy transfer(FRET)assay against the protease and polyphenols were identified as potential inhibitors using electrospray ionization-mass spectrometry(MS)/MS technique.Among different polyphenols,highly potent compounds were screened using molecular modeling approaches and consequently the most active compound was further evaluated against HCV NS4A-NS3 protease domain using FRET assay.RESULTS NS4A fused with NS3 protease domain gene was overexpressed and the purified protein yield was high in comparison to the lower yield of the full-length NS3 protein.Furthermore,in enzyme kinetic studies,NS4A fused with NS3 protease proved to be functionally active compared to full-length NS3.So it was concluded that co-factor NS4A fusion is essential for the purification of functionally active protease.FRET assay was developed and validated by the half maximal inhibitory concentration(IC50)values of commercially available inhibitors.Screening of citrus fruit extracts against the native purified fused NS4A-NS3 protease domain showed that the grapefruit mesocarp extract exhibits the highest percentage inhibition 91%of protease activity.Among the compounds identified by LCMS analysis,hesperidin showed strong binding affinity with the protease catalytic triad having S-score value of-10.98.CONCLUSION Fused NS4A-NS3 protease is functionally more active,which is effectively inhibited by hesperidin from the grapefruit mesocarp extract with an IC50 value of 23.32μmol/L.展开更多
A series of N1-substituted-3-aryl-4-alkyl-4, 5-dihydro-1H-1-pyrazolethiocarboxamide were prepared from the Mannich bases of aryl ketones in good yields. Some derivatives were found to be active against the cysteine p...A series of N1-substituted-3-aryl-4-alkyl-4, 5-dihydro-1H-1-pyrazolethiocarboxamide were prepared from the Mannich bases of aryl ketones in good yields. Some derivatives were found to be active against the cysteine protease of T.cruzi..展开更多
Background:Since the emergence of coronavirus disease 2019 to date,there is no available approved drug or definitive treatment for coronavirus disease 2019 viral infection,and the identification of novel hits against ...Background:Since the emergence of coronavirus disease 2019 to date,there is no available approved drug or definitive treatment for coronavirus disease 2019 viral infection,and the identification of novel hits against therapeutic targets has become a global emergency.Echinacea purpurea is a traditional herb utilized to treat cough,fever,sore throat,respiratory tract infection,and so on as an immune stimulant.In this study,in silico molecular docking approach was used to screen phytocompounds from E.purpurea against severe acute respiratory syndrome coronavirus 2 main protease 3C-like protease(3CLpro)and severe acute respiratory syndrome coronavirus main peptidase(96%sequence similarity)to blunt the viral gene expression and viral replication.Methods:Initially,we screened phytocompounds for their druggability and ADMET property.Furthermore,x-ray crystallographic structures of main proteases 3CLpro and main peptidase having Protein Data Bank ID 6LU7 and 2GTB were used as protein targets for the identification of potential drug candidates.We performed docking using AutoDock Vina by PyRx 0.8 software.BIOVIA Discovery Studio Visualizer v2019 was used to analyze ligand-protein complex.The probable protein targets of the selected compound were predicted by BindingDB(P≥0.7).STRING and Kyoto Encyclopedia of Genes and Genomes pathways are utilized to identify the molecular pathways modulated by the predicted targets(FDR≤0.05),and the network interaction between compounds and protein pathways was constructed by Cytoscape 3.6.1.Results:Among all the compounds,chlorogenic acid showed druggable characteristics and scored the lowest binding energy with main protease and main peptidase via interacting with active site 1 domain amino acid residues.Interestingly,chlorogenic acid interacted with Phe140 main protease 3CLpro,which is potentially involved in the dimerization.Enrichment analysis identified chlorogenic acid to modulate insulin resistance,necroptosis,interleukin-17,tumor necrosis factor signaling pathway,legionellosis,T helper 17 cell differentiation,advanced glycation end products and receptor for advanced glycation end products,mitogen-activated protein kinase,Ras,estrogen,vascular endothelial growth factor,B-cell receptor,nuclear factor kappa B,Rap1,hypoxia inducible factor-1,phosphatidylinositide 3-kinase-Akt,insulin,mechanistic target of rapamycin,p53,retinoic acid inducible gene I like receptor,and ErbB signaling pathways.Conclusion:Chlorogenic acid may act as a potent main protease 3CLpro inhibitor and may also inhibit the severe acute respiratory syndrome coronavirus 2 dimerization,viral gene expression,and replication within the lung epithelium.Chlorogenic acid may go a long way in finding one of the multipronged solutions to tackle coronavirus disease 2019 viral infection in the future.展开更多
Objective:To explore the effect of the protease inhibitor from Agaricus bisporus(J.E.Lange)Imbach(AbPI)on glucose uptake and oxidative stress in 3 T3-L1 adipocytes.Methods:Adipocytes were differentiated and stained wi...Objective:To explore the effect of the protease inhibitor from Agaricus bisporus(J.E.Lange)Imbach(AbPI)on glucose uptake and oxidative stress in 3 T3-L1 adipocytes.Methods:Adipocytes were differentiated and stained with OilRed-O staining to confirm adipogenesis.The toxic/protective effect of AbPI on the adipocytes was determined by MTT assay,intracellular reactive oxygen species generation through flow cytometry,and morphologically through confocal microscopy using propidium iodide,4,6-diamino-2-phenylindol dihydrochloride,and 2’,7’-dichlorofluorescein diacetate dyes.The uptake of fluorescent glucose analog,2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose by adipocytes was also studied through confocal microscopy.Results:MTT assay showed that the cell survival rate was(28.00±3.00)%,(92.33±2.60)%,and(71.34±2.10)%in the presence of 2 mM H2O2,AbPI alone,and AbPI and H2O2 both,respectively,in comparison to the control.Oil-Red-O staining indicated that Ab PI enhanced adipogenesis.AbPI stimulated the glucose uptake by adipocytes similar to the drug rosiglitazone,and showed insulinsensitizing effect in the presence of insulin,but failed to stimulate the uptake in the absence of insulin.Intracellular reactive oxygen species generation was reduced in differentiating adipocytes upon Ab PI treatment.Confocal microscopy showed that the damaged cell population rose to 3.50%,117.84%,and 261.50%in the presence of Ab PI alone,AbPI with H2O2,and H2O2 alone,respectively.Conclusions:The protease inhibitor enhances glucose uptake by adipocytes and exhibits a cytoprotective effect on them.展开更多
文摘BACKGROUND Hepatitis C virus genotype 3a(HCV G3a)is highly prevalent in Pakistan.Due to the elevated cost of available Food and Drug Administration-approved drugs against HCV,medicinal natural products of potent antiviral activity should be screened for the cost-effective treatment of the disease.Furthermore,from natural products,active compounds against vital HCV proteins like non-structural protein 3(NS3)protease could be identified to prevent viral proliferation in the host.AIM To develop cost-effective HCV genotype 3a NS3 protease inhibitors from citrus fruit extracts.METHODS Full-length NS3 without co-factor non-structural protein 4A(NS4A)and codon optimized NS3 protease in fusion with NS4A were expressed in Escherichia coli.The expressed protein was purified by metal ion affinity chromatography and gel filtration.Citrus fruit extracts were screened using fluorescence resonance energy transfer(FRET)assay against the protease and polyphenols were identified as potential inhibitors using electrospray ionization-mass spectrometry(MS)/MS technique.Among different polyphenols,highly potent compounds were screened using molecular modeling approaches and consequently the most active compound was further evaluated against HCV NS4A-NS3 protease domain using FRET assay.RESULTS NS4A fused with NS3 protease domain gene was overexpressed and the purified protein yield was high in comparison to the lower yield of the full-length NS3 protein.Furthermore,in enzyme kinetic studies,NS4A fused with NS3 protease proved to be functionally active compared to full-length NS3.So it was concluded that co-factor NS4A fusion is essential for the purification of functionally active protease.FRET assay was developed and validated by the half maximal inhibitory concentration(IC50)values of commercially available inhibitors.Screening of citrus fruit extracts against the native purified fused NS4A-NS3 protease domain showed that the grapefruit mesocarp extract exhibits the highest percentage inhibition 91%of protease activity.Among the compounds identified by LCMS analysis,hesperidin showed strong binding affinity with the protease catalytic triad having S-score value of-10.98.CONCLUSION Fused NS4A-NS3 protease is functionally more active,which is effectively inhibited by hesperidin from the grapefruit mesocarp extract with an IC50 value of 23.32μmol/L.
文摘A series of N1-substituted-3-aryl-4-alkyl-4, 5-dihydro-1H-1-pyrazolethiocarboxamide were prepared from the Mannich bases of aryl ketones in good yields. Some derivatives were found to be active against the cysteine protease of T.cruzi..
文摘Background:Since the emergence of coronavirus disease 2019 to date,there is no available approved drug or definitive treatment for coronavirus disease 2019 viral infection,and the identification of novel hits against therapeutic targets has become a global emergency.Echinacea purpurea is a traditional herb utilized to treat cough,fever,sore throat,respiratory tract infection,and so on as an immune stimulant.In this study,in silico molecular docking approach was used to screen phytocompounds from E.purpurea against severe acute respiratory syndrome coronavirus 2 main protease 3C-like protease(3CLpro)and severe acute respiratory syndrome coronavirus main peptidase(96%sequence similarity)to blunt the viral gene expression and viral replication.Methods:Initially,we screened phytocompounds for their druggability and ADMET property.Furthermore,x-ray crystallographic structures of main proteases 3CLpro and main peptidase having Protein Data Bank ID 6LU7 and 2GTB were used as protein targets for the identification of potential drug candidates.We performed docking using AutoDock Vina by PyRx 0.8 software.BIOVIA Discovery Studio Visualizer v2019 was used to analyze ligand-protein complex.The probable protein targets of the selected compound were predicted by BindingDB(P≥0.7).STRING and Kyoto Encyclopedia of Genes and Genomes pathways are utilized to identify the molecular pathways modulated by the predicted targets(FDR≤0.05),and the network interaction between compounds and protein pathways was constructed by Cytoscape 3.6.1.Results:Among all the compounds,chlorogenic acid showed druggable characteristics and scored the lowest binding energy with main protease and main peptidase via interacting with active site 1 domain amino acid residues.Interestingly,chlorogenic acid interacted with Phe140 main protease 3CLpro,which is potentially involved in the dimerization.Enrichment analysis identified chlorogenic acid to modulate insulin resistance,necroptosis,interleukin-17,tumor necrosis factor signaling pathway,legionellosis,T helper 17 cell differentiation,advanced glycation end products and receptor for advanced glycation end products,mitogen-activated protein kinase,Ras,estrogen,vascular endothelial growth factor,B-cell receptor,nuclear factor kappa B,Rap1,hypoxia inducible factor-1,phosphatidylinositide 3-kinase-Akt,insulin,mechanistic target of rapamycin,p53,retinoic acid inducible gene I like receptor,and ErbB signaling pathways.Conclusion:Chlorogenic acid may act as a potent main protease 3CLpro inhibitor and may also inhibit the severe acute respiratory syndrome coronavirus 2 dimerization,viral gene expression,and replication within the lung epithelium.Chlorogenic acid may go a long way in finding one of the multipronged solutions to tackle coronavirus disease 2019 viral infection in the future.
文摘Objective:To explore the effect of the protease inhibitor from Agaricus bisporus(J.E.Lange)Imbach(AbPI)on glucose uptake and oxidative stress in 3 T3-L1 adipocytes.Methods:Adipocytes were differentiated and stained with OilRed-O staining to confirm adipogenesis.The toxic/protective effect of AbPI on the adipocytes was determined by MTT assay,intracellular reactive oxygen species generation through flow cytometry,and morphologically through confocal microscopy using propidium iodide,4,6-diamino-2-phenylindol dihydrochloride,and 2’,7’-dichlorofluorescein diacetate dyes.The uptake of fluorescent glucose analog,2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose by adipocytes was also studied through confocal microscopy.Results:MTT assay showed that the cell survival rate was(28.00±3.00)%,(92.33±2.60)%,and(71.34±2.10)%in the presence of 2 mM H2O2,AbPI alone,and AbPI and H2O2 both,respectively,in comparison to the control.Oil-Red-O staining indicated that Ab PI enhanced adipogenesis.AbPI stimulated the glucose uptake by adipocytes similar to the drug rosiglitazone,and showed insulinsensitizing effect in the presence of insulin,but failed to stimulate the uptake in the absence of insulin.Intracellular reactive oxygen species generation was reduced in differentiating adipocytes upon Ab PI treatment.Confocal microscopy showed that the damaged cell population rose to 3.50%,117.84%,and 261.50%in the presence of Ab PI alone,AbPI with H2O2,and H2O2 alone,respectively.Conclusions:The protease inhibitor enhances glucose uptake by adipocytes and exhibits a cytoprotective effect on them.
