OBJECTIVE To investigate the structural requirements for effective binding of andrographolide(AGP)and its derivatives(SRJ09and SRJ23)to mutant K-ras for inhibition of exchange factor binding viain silico docking simul...OBJECTIVE To investigate the structural requirements for effective binding of andrographolide(AGP)and its derivatives(SRJ09and SRJ23)to mutant K-ras for inhibition of exchange factor binding viain silico docking simulations.METHODS The molecular docking studies were carried out by using SiteMap v3.4andGlide v6.6modules(Schrdinger,Inc.).Surface mapping on the 3-D X-ray crystal structures of three mutant K-ras proteins-K-rasG12V(PDB ID:4EPX),K-rasG12C(PDB ID:4LDJ),and K-rasG12D(PDB ID:4DSU),as well as wild-type K-ras protein(PDB ID:4LPK),was performed to generate possible sites for ligand binding.Thirty conformers were generated for each of the studied compounds,and these conformers were docked into each possible binding site in both wild-type and mutant K-ras proteins.The free energy of binding of the compounds with the wild-type and mutant K-ras proteins was performed using prime molecular mechanics with generalized Born and solvent accessibility(MM-GBSA)approach.RESULTS The conformers of AGP,SRJ09 and SRJ23that were found to form the most stable complex inside each possible binding siteas indicated by the highest binding free energy,both in wild-type and mutant proteins,were selected.A common binding site between switchⅠ and Ⅱregions,where a pocket surrounded by amino acid residues Lys5,Leu6,Val7,Ser39,Asp54,Leu56,Tyr71,Thr74,and Gly75,was found in all K-rasG12 mutants.This site corresponds to the hydrophobic binding pockets having aliphatic side-chain portionsas found previously for other Ras binders,which are located betweenα-helix 2 and the core β-sheets(between switchⅠ and Ⅱregions).This common binding pocket was not observed in the wild-type K-ras.A binding pocket adjacent to switchⅡregion(amino acid 60-72),where all ligands bind well,was found instead.All compoundsanchor well inside the common binding pocket in each of the K-fasG12 mutants and these compounds showed the strongest binding interactions to K-fasG12 C.SRJ09 and SRJ23 showed stronger binding interactions to both wild-type and mutant K-ras proteins as compared with the parent compound.Overall,the compounds displayed higher binding energies toall three mutant proteins as compared to their wild-type counterpart.CONCLUSION AGP,SRJ09,and SRJ23 are potential K-ras-targeting anti-cancer agents.The compounds target both wild-type and mutant K-ras but they bind to a different binding pocket in the wild-type protein.Both binding pockets found in wild-type and mutant K-ras involve switchⅡ region that binds the guanine nucleotide exchange factor(GEF)such as Son of Sevenless.These suggest a possible inhibition of exchange factor binding to both wild-type and mutant K-ras proteins.Lower binding energies of the compounds to wild-type K-ras protein suggest a transient binding and inhibition.Stronger binding of all compounds to mutant K-ras proteins could lead to more targeted and prolonged inhibition.展开更多
OBJECTIVE Steroid-resistant airway hyper-responsiveness(AHR)has been proposed to be related to the activation of innate host defense pathways such as those induced by LPS,IFN-γ,and LPS/IFN-γ-stimulated essential med...OBJECTIVE Steroid-resistant airway hyper-responsiveness(AHR)has been proposed to be related to the activation of innate host defense pathways such as those induced by LPS,IFN-γ,and LPS/IFN-γ-stimulated essential mediator IL-27.We investigated whether andrographolide,apreviously demonstrated anti-inflammatory bioactive molecule extracted from the plant Andrographis paniculata,could restore steroid sensitivity to block LPS/IFN-γ-induced IL-27 production and AHR viaits anti-oxidative property.METHODS Mouse macrophage cell line Raw264.7,mouse primary pulmonary monocyte/macrophage,and BALB/c mouse were treated with LPS/IFN-γ,in the presence and absence of increasing doses of dexamethasone and/or andrographolide.mRNA and protein levels of IL-27 in vitro and in vivo were examined,and mouse AHR was assessed.RESULTS Dexamethasone alone failed to inhibit LPS/IFN-γ-induced IL-27 and AHR in mice.Andrographolide significantly facilitated the suppressive effect of dexamethasone on LPS/IFN-γ-induced IL-27 level in macrophage cell line and primary monocyte/macrophage,mouse bronchoalveolar lavage fluid and lung tissue,and furthermore on the incurring AHR.LPS/IFN-γdid not impede nuclear translocation of glucocorticoid receptors but diminishthe protein level of histone deacetylase-2(HDAC2),an essential epigenetic enzymeresponsible for steroid anti-inflammatory action.Andrographolide at low doses(5μmol·L-1 in vitro;1mg·kg-1,ip in vivo)restored nuclear HDAC2 protein levels both in cells and in mouse lungs,possibly via suppression of PI3K/Akt signaling pathway and up-regulation of the anti-oxidative transcription factor Nrf-2level.CONCLUSION Our data suggest that andrographolide may resensitize steroid action on blocking LPS/IFN-γ-induced IL-27 and resultant AHR by restoring HDAC2 level.展开更多
OBJECTIVE To develop a 2-week cigarette smoke(CS)acute lung injury model exacerbated by haemophilus influenzae(NTHi)and study the protective effect of andrographolide in this COPD model.METHODS Female BALB/c mice,6-8-...OBJECTIVE To develop a 2-week cigarette smoke(CS)acute lung injury model exacerbated by haemophilus influenzae(NTHi)and study the protective effect of andrographolide in this COPD model.METHODS Female BALB/c mice,6-8-week-old,were exposed to 4% 3R4 FCS delivered using aperistaltic pump daily for 2 weeks to induce an acute lung injury model.After 2 weeks of smoking,mice were inoculated intratracheally with NTHi to induce exacerbation on the model.Mice were sacrificed 48 h after last bacteria challenge and lung samples were collected for various analyses.RESULTS After developing a 2-week CS acute lung injury model exacerbated by NTHi,the CS+NTHi group was shown to have a higher inflammatory response,higher bacterial clearance,an upregulation of MMP12 mRNA levels and decrease in TIMP1 mRNA levels in the lungs.Administration of Andrographolide suppressed BALF lung cellular infiltrates,TNF-α,CXCL1/KC,IL-1βand 8-OHdG protein levels,together with increased HO-1 and GR mRNA levels and decreased MMP-8 and MMP-9 mRNA levels.Andrographolide was able to ameliorate lung histopathology as observed with H&E staining and inflammation scoring.Andrographolide was also shown to reduce Keap-1 level in lungs without affecting DJ-1 level.CONCLUSION This study demonstrates the protective effect of andrographolide in a novel 2-week CS acute lung injury model exacerbated by NTHi and presents it as a potential therapeutic for COPD.展开更多
OBJECTIVE To analyse structure and relationship of several andrographolide derivatives in multiple in vivo and in vitro angiogenesis assays,and to demonstrate a novel compound named AGL-2as a potential anti-angiogenes...OBJECTIVE To analyse structure and relationship of several andrographolide derivatives in multiple in vivo and in vitro angiogenesis assays,and to demonstrate a novel compound named AGL-2as a potential anti-angiogenesis agent.METHODS Human umbilical vein endothelial cells(HUVECs)in vitro and zebrafish(Danio rerio)in vivo models were used to screen and identify the anti-angiogenesis activities of six andrographolide derivatives;namely,AGL-1,AGL-2,AGS-72,AGS-72 a,AGS-79 and AGP-151.RESULTS AGL-2exhibited the strongest anti-angiogenic activity among all the derivatives in zebrafish model.Interestingly,another compound named AGS-72 showed stronger anti-angiogenic activity than AGL-2 in VEGF-induced HUVECs proliferation,migration,invasion and tube formation assays.In addition,AGL-2 was found to suppress the VEGF-induced VEGFR-2auto-phosphorylation and inhibit the activity of VEGFR-2 mediated signaling cascades in a dose-dependent manner.CONCLUSION AGL-2was demonstrated to be a promising anti-angiogenic agent among all the tested derivatives.The mechanism underlying the anti-angiogenic activity of AGL-2 probably involve VEGFR-2 signaling pathway.Even though,how some of chemical structure alterations result in discrepancy between in vivo and in vitro activities still remains to be resolved,this study shall provide new insight into how modification of the chemical structure of andrographolide affects this newly identified anti-angiogenesis activity.Meanwhile,AGL-2 can be exploited as a potential therapeutic agent for the treatment of angiogenesis-related diseases.展开更多
We embarked on the discovery of anticancer agents from andrographolide nearly 15 years ago.Thus far,a few lead semisynthetic compounds have been identified,but only recently we managed to pinpoint their potential mole...We embarked on the discovery of anticancer agents from andrographolide nearly 15 years ago.Thus far,a few lead semisynthetic compounds have been identified,but only recently we managed to pinpoint their potential molecular target.Through in silico and cell-based studies,these lead molecules have been found to bind K-ras oncoprotein and disrupt its function.Further molecular docking analysis suggested the compounds targeted both wild-type and oncogenic mutant K-ras.However,the binding affinity was greater for the oncogenic protein.Low binding energies to wild-type K-ras protein suggested transient binding and inhibition.The compounds showed stronger binding interactions to all three mutant K-ras proteins(G12V,G12 Cand G12D)with average free energies(ΔG bind)of-82kcal·mol-1 as compared with-61kcal·mol-1 for the wild-type protein.It is noteworthy that the binding pocket in wild-type K-ras protein,however,is different from that of the mutant proteins.SRJ23,one of the lead compounds,showed the strongest binding interactions to all three mutant K-ras proteins.Stronger binding to the mutant proteins could lead to more targeted and prolonged inhibition.Investigation into the effect of the compounds on RAS-MAPK pathway showed this pathway was disrupted in colon,breast and prostate cancer cells.In vivo studies revealed the compounds retarded the growth of human colon(HCT-116)and prostate(PC-3)cancer xenografts in mice.All of the above prompted us to synthesise derivatives of the lead compounds for improvement of binding affinity for the oncogenic K-ras.A preliminary in silico exploration found some compounds with such property and these compounds are presently undergoing extensive pharmacological investigations.展开更多
Andrographis paniculata contains two main diterpenoid constituents,andrographolide(AGP)and 14-deoxy-11,12-didehydroandrographolide(DDAG),which were found to exhibit antiasthma effects in a mouse asthma model.However,d...Andrographis paniculata contains two main diterpenoid constituents,andrographolide(AGP)and 14-deoxy-11,12-didehydroandrographolide(DDAG),which were found to exhibit antiasthma effects in a mouse asthma model.However,due to inadequacies of both compounds in terms of drug-likeness,DDAG analogues were semisynthesised to tackle these shortcomings.The objective of the study was to investigate the potential of DDAG analogues as new antiasthma agents.Among the analogues,(SRS27)was proven to inhibit cysteinyl leukotrienes(CysLT)and nitric oxide(NO)synthesis in mouse macrophages,like AGP.DDAG,on the other hand,failed to exhibit such activities.SRS27 is less cytotoxic than AGP,suggests that a simple chemical modification of DDAG produces a compound with CysLT and NO inhibitory activites similar to AGP while maintaining toxicity profile similar to DDAG.It is interesting to note that other analogues such as SRS28,SRS49,SRS76 and SRS83 with chemical modifications on the same carbon numbers 3 and 19 of DDAG were unable to show inhibition of CysLT and NO synthesis.Consequently,the potential anti-inflammatory effect of SRS27 was investigated in ovalbumin(OVA)-induced mouse asthma model.The compound was administered in a prophylactic manner and showed a substantial decrease in mouse asthma model parameters.SRS27 at 3mg·kg-1 significantly reduced OVA-induced total cell such as macrophages,eosinophils,lymphocytes and neutrophils,as well as inflammatory cytokines such as IL-4,IL-5,IL-13 and eotaxin in bronchoalveolar lavage BAL fluid.The compound also suppressed serum IgE production.In addition,SRS27 suppressed mucus hyper-secretion and expression of inflammatory mediators such as TNF-α,MCP-1,Muc5 ac,RANTES,IL-33 and iNOS.SRS27 is the first known DDAG derivative tested positive in mouse asthma model and as such SRS27 could serve as a prototype prophylactic agent.展开更多
基金The project supported by Ministry of Education,Malaysia(Research University Grant Scheme Grant 04-02-12-2017RU)
文摘OBJECTIVE To investigate the structural requirements for effective binding of andrographolide(AGP)and its derivatives(SRJ09and SRJ23)to mutant K-ras for inhibition of exchange factor binding viain silico docking simulations.METHODS The molecular docking studies were carried out by using SiteMap v3.4andGlide v6.6modules(Schrdinger,Inc.).Surface mapping on the 3-D X-ray crystal structures of three mutant K-ras proteins-K-rasG12V(PDB ID:4EPX),K-rasG12C(PDB ID:4LDJ),and K-rasG12D(PDB ID:4DSU),as well as wild-type K-ras protein(PDB ID:4LPK),was performed to generate possible sites for ligand binding.Thirty conformers were generated for each of the studied compounds,and these conformers were docked into each possible binding site in both wild-type and mutant K-ras proteins.The free energy of binding of the compounds with the wild-type and mutant K-ras proteins was performed using prime molecular mechanics with generalized Born and solvent accessibility(MM-GBSA)approach.RESULTS The conformers of AGP,SRJ09 and SRJ23that were found to form the most stable complex inside each possible binding siteas indicated by the highest binding free energy,both in wild-type and mutant proteins,were selected.A common binding site between switchⅠ and Ⅱregions,where a pocket surrounded by amino acid residues Lys5,Leu6,Val7,Ser39,Asp54,Leu56,Tyr71,Thr74,and Gly75,was found in all K-rasG12 mutants.This site corresponds to the hydrophobic binding pockets having aliphatic side-chain portionsas found previously for other Ras binders,which are located betweenα-helix 2 and the core β-sheets(between switchⅠ and Ⅱregions).This common binding pocket was not observed in the wild-type K-ras.A binding pocket adjacent to switchⅡregion(amino acid 60-72),where all ligands bind well,was found instead.All compoundsanchor well inside the common binding pocket in each of the K-fasG12 mutants and these compounds showed the strongest binding interactions to K-fasG12 C.SRJ09 and SRJ23 showed stronger binding interactions to both wild-type and mutant K-ras proteins as compared with the parent compound.Overall,the compounds displayed higher binding energies toall three mutant proteins as compared to their wild-type counterpart.CONCLUSION AGP,SRJ09,and SRJ23 are potential K-ras-targeting anti-cancer agents.The compounds target both wild-type and mutant K-ras but they bind to a different binding pocket in the wild-type protein.Both binding pockets found in wild-type and mutant K-ras involve switchⅡ region that binds the guanine nucleotide exchange factor(GEF)such as Son of Sevenless.These suggest a possible inhibition of exchange factor binding to both wild-type and mutant K-ras proteins.Lower binding energies of the compounds to wild-type K-ras protein suggest a transient binding and inhibition.Stronger binding of all compounds to mutant K-ras proteins could lead to more targeted and prolonged inhibition.
基金The project supported by National Medical Research Council of Singapore(NMRC/CBRG/0027/2012)
文摘OBJECTIVE Steroid-resistant airway hyper-responsiveness(AHR)has been proposed to be related to the activation of innate host defense pathways such as those induced by LPS,IFN-γ,and LPS/IFN-γ-stimulated essential mediator IL-27.We investigated whether andrographolide,apreviously demonstrated anti-inflammatory bioactive molecule extracted from the plant Andrographis paniculata,could restore steroid sensitivity to block LPS/IFN-γ-induced IL-27 production and AHR viaits anti-oxidative property.METHODS Mouse macrophage cell line Raw264.7,mouse primary pulmonary monocyte/macrophage,and BALB/c mouse were treated with LPS/IFN-γ,in the presence and absence of increasing doses of dexamethasone and/or andrographolide.mRNA and protein levels of IL-27 in vitro and in vivo were examined,and mouse AHR was assessed.RESULTS Dexamethasone alone failed to inhibit LPS/IFN-γ-induced IL-27 and AHR in mice.Andrographolide significantly facilitated the suppressive effect of dexamethasone on LPS/IFN-γ-induced IL-27 level in macrophage cell line and primary monocyte/macrophage,mouse bronchoalveolar lavage fluid and lung tissue,and furthermore on the incurring AHR.LPS/IFN-γdid not impede nuclear translocation of glucocorticoid receptors but diminishthe protein level of histone deacetylase-2(HDAC2),an essential epigenetic enzymeresponsible for steroid anti-inflammatory action.Andrographolide at low doses(5μmol·L-1 in vitro;1mg·kg-1,ip in vivo)restored nuclear HDAC2 protein levels both in cells and in mouse lungs,possibly via suppression of PI3K/Akt signaling pathway and up-regulation of the anti-oxidative transcription factor Nrf-2level.CONCLUSION Our data suggest that andrographolide may resensitize steroid action on blocking LPS/IFN-γ-induced IL-27 and resultant AHR by restoring HDAC2 level.
基金The project supported by National Medical Research Council(NMRC/CBRG/0027/2012)
文摘OBJECTIVE To develop a 2-week cigarette smoke(CS)acute lung injury model exacerbated by haemophilus influenzae(NTHi)and study the protective effect of andrographolide in this COPD model.METHODS Female BALB/c mice,6-8-week-old,were exposed to 4% 3R4 FCS delivered using aperistaltic pump daily for 2 weeks to induce an acute lung injury model.After 2 weeks of smoking,mice were inoculated intratracheally with NTHi to induce exacerbation on the model.Mice were sacrificed 48 h after last bacteria challenge and lung samples were collected for various analyses.RESULTS After developing a 2-week CS acute lung injury model exacerbated by NTHi,the CS+NTHi group was shown to have a higher inflammatory response,higher bacterial clearance,an upregulation of MMP12 mRNA levels and decrease in TIMP1 mRNA levels in the lungs.Administration of Andrographolide suppressed BALF lung cellular infiltrates,TNF-α,CXCL1/KC,IL-1βand 8-OHdG protein levels,together with increased HO-1 and GR mRNA levels and decreased MMP-8 and MMP-9 mRNA levels.Andrographolide was able to ameliorate lung histopathology as observed with H&E staining and inflammation scoring.Andrographolide was also shown to reduce Keap-1 level in lungs without affecting DJ-1 level.CONCLUSION This study demonstrates the protective effect of andrographolide in a novel 2-week CS acute lung injury model exacerbated by NTHi and presents it as a potential therapeutic for COPD.
基金The project supported by Science and Technology Development Fund of Macao SAR(078/2011/A3)Research Committee of University of Macao〔MYRG138(Y1-Y4)-ICMS12-LMY〕
文摘OBJECTIVE To analyse structure and relationship of several andrographolide derivatives in multiple in vivo and in vitro angiogenesis assays,and to demonstrate a novel compound named AGL-2as a potential anti-angiogenesis agent.METHODS Human umbilical vein endothelial cells(HUVECs)in vitro and zebrafish(Danio rerio)in vivo models were used to screen and identify the anti-angiogenesis activities of six andrographolide derivatives;namely,AGL-1,AGL-2,AGS-72,AGS-72 a,AGS-79 and AGP-151.RESULTS AGL-2exhibited the strongest anti-angiogenic activity among all the derivatives in zebrafish model.Interestingly,another compound named AGS-72 showed stronger anti-angiogenic activity than AGL-2 in VEGF-induced HUVECs proliferation,migration,invasion and tube formation assays.In addition,AGL-2 was found to suppress the VEGF-induced VEGFR-2auto-phosphorylation and inhibit the activity of VEGFR-2 mediated signaling cascades in a dose-dependent manner.CONCLUSION AGL-2was demonstrated to be a promising anti-angiogenic agent among all the tested derivatives.The mechanism underlying the anti-angiogenic activity of AGL-2 probably involve VEGFR-2 signaling pathway.Even though,how some of chemical structure alterations result in discrepancy between in vivo and in vitro activities still remains to be resolved,this study shall provide new insight into how modification of the chemical structure of andrographolide affects this newly identified anti-angiogenesis activity.Meanwhile,AGL-2 can be exploited as a potential therapeutic agent for the treatment of angiogenesis-related diseases.
文摘We embarked on the discovery of anticancer agents from andrographolide nearly 15 years ago.Thus far,a few lead semisynthetic compounds have been identified,but only recently we managed to pinpoint their potential molecular target.Through in silico and cell-based studies,these lead molecules have been found to bind K-ras oncoprotein and disrupt its function.Further molecular docking analysis suggested the compounds targeted both wild-type and oncogenic mutant K-ras.However,the binding affinity was greater for the oncogenic protein.Low binding energies to wild-type K-ras protein suggested transient binding and inhibition.The compounds showed stronger binding interactions to all three mutant K-ras proteins(G12V,G12 Cand G12D)with average free energies(ΔG bind)of-82kcal·mol-1 as compared with-61kcal·mol-1 for the wild-type protein.It is noteworthy that the binding pocket in wild-type K-ras protein,however,is different from that of the mutant proteins.SRJ23,one of the lead compounds,showed the strongest binding interactions to all three mutant K-ras proteins.Stronger binding to the mutant proteins could lead to more targeted and prolonged inhibition.Investigation into the effect of the compounds on RAS-MAPK pathway showed this pathway was disrupted in colon,breast and prostate cancer cells.In vivo studies revealed the compounds retarded the growth of human colon(HCT-116)and prostate(PC-3)cancer xenografts in mice.All of the above prompted us to synthesise derivatives of the lead compounds for improvement of binding affinity for the oncogenic K-ras.A preliminary in silico exploration found some compounds with such property and these compounds are presently undergoing extensive pharmacological investigations.
基金The project supported by grant(BMRC/09/1/21/19/595)from the BioMedical Research Council of SingaporeResearch University Grant Scheme(RUGS)(04-02-12-2017RU)from the Ministry of Higher Education of Malaysia
文摘Andrographis paniculata contains two main diterpenoid constituents,andrographolide(AGP)and 14-deoxy-11,12-didehydroandrographolide(DDAG),which were found to exhibit antiasthma effects in a mouse asthma model.However,due to inadequacies of both compounds in terms of drug-likeness,DDAG analogues were semisynthesised to tackle these shortcomings.The objective of the study was to investigate the potential of DDAG analogues as new antiasthma agents.Among the analogues,(SRS27)was proven to inhibit cysteinyl leukotrienes(CysLT)and nitric oxide(NO)synthesis in mouse macrophages,like AGP.DDAG,on the other hand,failed to exhibit such activities.SRS27 is less cytotoxic than AGP,suggests that a simple chemical modification of DDAG produces a compound with CysLT and NO inhibitory activites similar to AGP while maintaining toxicity profile similar to DDAG.It is interesting to note that other analogues such as SRS28,SRS49,SRS76 and SRS83 with chemical modifications on the same carbon numbers 3 and 19 of DDAG were unable to show inhibition of CysLT and NO synthesis.Consequently,the potential anti-inflammatory effect of SRS27 was investigated in ovalbumin(OVA)-induced mouse asthma model.The compound was administered in a prophylactic manner and showed a substantial decrease in mouse asthma model parameters.SRS27 at 3mg·kg-1 significantly reduced OVA-induced total cell such as macrophages,eosinophils,lymphocytes and neutrophils,as well as inflammatory cytokines such as IL-4,IL-5,IL-13 and eotaxin in bronchoalveolar lavage BAL fluid.The compound also suppressed serum IgE production.In addition,SRS27 suppressed mucus hyper-secretion and expression of inflammatory mediators such as TNF-α,MCP-1,Muc5 ac,RANTES,IL-33 and iNOS.SRS27 is the first known DDAG derivative tested positive in mouse asthma model and as such SRS27 could serve as a prototype prophylactic agent.
