Human serum albumin （HSA） is a plasma protein responsible for the binding and transport of fatty acids and a variety of exogenous chemicals such as drugs and environmental pollutants. Such binding plays a crucial ro...Human serum albumin （HSA） is a plasma protein responsible for the binding and transport of fatty acids and a variety of exogenous chemicals such as drugs and environmental pollutants. Such binding plays a crucial role in determining the ADME （absorption, distribution, metabolism, and excretion） and bioavailability of the pollutants. The binding interaction between HSA and acetic acid （C2）, octanoic acid （C8） and dodecanoic acid （C12） has been investigated by the combination of site-specific fluorescent probe, tryptophan intrinsic fluorescence and tyrosine electrochemistry. For the study of the fatty acid interaction with the two drug-binding sites on HSA, two fluorescent probes, dansylamide and dansyl-L-proline were employed in the displacement measurements. Intrinsic fluorescence of tryptophan in HSA was monitored upon addition of the fatty acids into HSA. Electrocatalyzed response of the tyrosine residues in HSA by a redox mediator was used to investigate the binding interaction. Qualitatively, observations from these three approaches were very similar. HSA did not show any change in the fluorescence and electrochemical experiments after mixing with C2, suggesting there is no significant interaction with the short-chain fatty acid. For C8, the measured signal dropped in a single-exponential mode, indicating an independent and non-cooperative binding. The calculated association constant and binding ratio were 3.1 × 10^6 L/mol and 1 with drug binding Site Ⅰ, 1.1 × 107 L/mol and 1 with Site Ⅱ, and 7.0× 0^4 L/mol and 4 with the tryptophan site, respectively. The measurements with C12 displayed multiple phases of fluorescence change, suggesting cooperativity and allosteric effect of the C12 binding. These results correlate well with those obtained by the established methods, and validate the new approach as a viable tool to study the interactions of environmental pollutants with biological molecules.展开更多
Breast cancer is presently one of the most common malignancies worldwide,with a higher fatality rate.In this study,a quantitative structure-activity relationship(QSAR)model of compound biological activity and ADMET(Ab...Breast cancer is presently one of the most common malignancies worldwide,with a higher fatality rate.In this study,a quantitative structure-activity relationship(QSAR)model of compound biological activity and ADMET(Absorption,Distribution,Metabolism,Excretion,Toxicity)properties prediction model were performed using estrogen receptor alpha(ERα)antagonist information collected from compound samples.We first utilized grey relation analysis(GRA)in conjunction with the random forest(RF)algorithm to identify the top 20 molecular descriptor variables that have the greatest influence on biological activity,and then we used Spearman correlation analysis to identify 16 independent variables.Second,a QSAR model of the compound were developed based on BP neural network(BPNN),genetic algorithm optimized BP neural network(GA-BPNN),and support vector regression(SVR).The BPNN,the SVR,and the logistic regression(LR)models were then used to identify and predict the ADMET properties of substances,with the prediction impacts of each model compared and assessed.The results reveal that a SVR model was used in QSAR quantitative prediction,and in the classification prediction of ADMET properties:the SVR model predicts the Caco-2 and hERG(human Ether-a-go-go Related Gene)properties,the LR model predicts the cytochrome P450 enzyme 3A4 subtype(CYP3A4)and Micronucleus(MN)properties,and the BPNN model predicts the Human Oral Bioavailability(HOB)properties.Finally,information entropy theory is used to validate the rationality of variable screening,and sensitivity analysis of the model demonstrates that the constructed model has high accuracy and stability,which can be used as a reference for screening probable active compounds and drug discovery.展开更多
The human body consists of several physiological barriers that express a number of membrane transporters. For an orally absorbed drug the intestinal, hepatic, renal and blood-brain barriers are of the greatest importa...The human body consists of several physiological barriers that express a number of membrane transporters. For an orally absorbed drug the intestinal, hepatic, renal and blood-brain barriers are of the greatest importance. The ATP-binding cassette(ABC) transporters that mediate cellular efflux and the solute carrier transporters that mostly mediate cellular uptake are the two superfamilies responsible for membrane transport of vast majority of drugs and drug metabolites. The total number of human transporters in the two superfamilies exceeds 400, and about 40-50 transporters have been characterized for drug transport. The latest Food and Drug Administration guidance focuses on P-glycoprotein, breast cancer resistance protein, organic anion transporting polypeptide 1B1(OATP1B1), OATP1B3, organic cation transporter 2(OCT2), and organic anion transporters 1(OAT1) and OAT3. The European Medicines Agency's shortlist additionally contains the bile salt export pump, OCT1, and the multidrug and toxin extrusion transporters, multidrug and toxin ex-trusion protein 1(MATE1) and MATE2/MATE2 K. A variety of transporter assays are available to test drugtransporter interactions, transporter-mediated drugdrug interactions, and transporter-mediated toxicity. The drug binding site of ABC transporters is accessible from the cytoplasm or the inner leaflet of the plasma membrane. Therefore, vesicular transport assays utilizing inside-out vesicles are commonly used assays, where the directionality of transport results in drugs being transported into the vesicle. Monolayer assays utilizing polarized cells expressing efflux transporters are the test systems suggested by regulatory agencies. However, in some monolayers, uptake transporters must be coexpressed with efflux transporters to assure detectable transport of low passive permeability drugs. For uptake transporters mediating cellular drug uptake, utilization of stable transfectants have been suggested. In vivo animal models complete the testing battery. Some issues, such as in vivo relevance, gender difference, age and ontogeny issues can only be addressed using in vivo models. Transporter specificity is provided by using knock-out or mutant models. Alternatively, chemical knock-outs can be employed. Compensatory changes are less likely when using chemical knockouts. On the other hand, specific inhibitors for some uptake transporters are not available, limiting the options to genetic knock-outs.展开更多
基金supported by the National Basic Re-search Program of China (No. 2006CB403303)the Knowledge Innovation Program of the Chinese Academy of Sciences (No. KZCX2-YW-420-1)the National Natural Science Foundation of China (No. 20890112)
文摘Human serum albumin （HSA） is a plasma protein responsible for the binding and transport of fatty acids and a variety of exogenous chemicals such as drugs and environmental pollutants. Such binding plays a crucial role in determining the ADME （absorption, distribution, metabolism, and excretion） and bioavailability of the pollutants. The binding interaction between HSA and acetic acid （C2）, octanoic acid （C8） and dodecanoic acid （C12） has been investigated by the combination of site-specific fluorescent probe, tryptophan intrinsic fluorescence and tyrosine electrochemistry. For the study of the fatty acid interaction with the two drug-binding sites on HSA, two fluorescent probes, dansylamide and dansyl-L-proline were employed in the displacement measurements. Intrinsic fluorescence of tryptophan in HSA was monitored upon addition of the fatty acids into HSA. Electrocatalyzed response of the tyrosine residues in HSA by a redox mediator was used to investigate the binding interaction. Qualitatively, observations from these three approaches were very similar. HSA did not show any change in the fluorescence and electrochemical experiments after mixing with C2, suggesting there is no significant interaction with the short-chain fatty acid. For C8, the measured signal dropped in a single-exponential mode, indicating an independent and non-cooperative binding. The calculated association constant and binding ratio were 3.1 × 10^6 L/mol and 1 with drug binding Site Ⅰ, 1.1 × 107 L/mol and 1 with Site Ⅱ, and 7.0× 0^4 L/mol and 4 with the tryptophan site, respectively. The measurements with C12 displayed multiple phases of fluorescence change, suggesting cooperativity and allosteric effect of the C12 binding. These results correlate well with those obtained by the established methods, and validate the new approach as a viable tool to study the interactions of environmental pollutants with biological molecules.
基金Supported by the Postgraduate Research&Practice Innovation Program of Jiangsu Province(KYCX23_0082)
文摘Breast cancer is presently one of the most common malignancies worldwide,with a higher fatality rate.In this study,a quantitative structure-activity relationship(QSAR)model of compound biological activity and ADMET(Absorption,Distribution,Metabolism,Excretion,Toxicity)properties prediction model were performed using estrogen receptor alpha(ERα)antagonist information collected from compound samples.We first utilized grey relation analysis(GRA)in conjunction with the random forest(RF)algorithm to identify the top 20 molecular descriptor variables that have the greatest influence on biological activity,and then we used Spearman correlation analysis to identify 16 independent variables.Second,a QSAR model of the compound were developed based on BP neural network(BPNN),genetic algorithm optimized BP neural network(GA-BPNN),and support vector regression(SVR).The BPNN,the SVR,and the logistic regression(LR)models were then used to identify and predict the ADMET properties of substances,with the prediction impacts of each model compared and assessed.The results reveal that a SVR model was used in QSAR quantitative prediction,and in the classification prediction of ADMET properties:the SVR model predicts the Caco-2 and hERG(human Ether-a-go-go Related Gene)properties,the LR model predicts the cytochrome P450 enzyme 3A4 subtype(CYP3A4)and Micronucleus(MN)properties,and the BPNN model predicts the Human Oral Bioavailability(HOB)properties.Finally,information entropy theory is used to validate the rationality of variable screening,and sensitivity analysis of the model demonstrates that the constructed model has high accuracy and stability,which can be used as a reference for screening probable active compounds and drug discovery.
基金Supported by FP7 IMI MIP-DILI:Mechanism-based integrated systems for the prediction of drug-induced liver injuryFP7 Eustroke,Health-F2-2008-202213:European Stroke Research Network+1 种基金TUDAS-1-2006-0029,OMFB-00505/2007:Development of HTS kit for analyzing transporter-drug interactions using cholesterol treated insect-cells expressing human MXR transporterGOP-1.1.1-11-2011-0017:Integrated preclinical tools for the determination and the enhancement of drug absorption
文摘The human body consists of several physiological barriers that express a number of membrane transporters. For an orally absorbed drug the intestinal, hepatic, renal and blood-brain barriers are of the greatest importance. The ATP-binding cassette(ABC) transporters that mediate cellular efflux and the solute carrier transporters that mostly mediate cellular uptake are the two superfamilies responsible for membrane transport of vast majority of drugs and drug metabolites. The total number of human transporters in the two superfamilies exceeds 400, and about 40-50 transporters have been characterized for drug transport. The latest Food and Drug Administration guidance focuses on P-glycoprotein, breast cancer resistance protein, organic anion transporting polypeptide 1B1(OATP1B1), OATP1B3, organic cation transporter 2(OCT2), and organic anion transporters 1(OAT1) and OAT3. The European Medicines Agency's shortlist additionally contains the bile salt export pump, OCT1, and the multidrug and toxin extrusion transporters, multidrug and toxin ex-trusion protein 1(MATE1) and MATE2/MATE2 K. A variety of transporter assays are available to test drugtransporter interactions, transporter-mediated drugdrug interactions, and transporter-mediated toxicity. The drug binding site of ABC transporters is accessible from the cytoplasm or the inner leaflet of the plasma membrane. Therefore, vesicular transport assays utilizing inside-out vesicles are commonly used assays, where the directionality of transport results in drugs being transported into the vesicle. Monolayer assays utilizing polarized cells expressing efflux transporters are the test systems suggested by regulatory agencies. However, in some monolayers, uptake transporters must be coexpressed with efflux transporters to assure detectable transport of low passive permeability drugs. For uptake transporters mediating cellular drug uptake, utilization of stable transfectants have been suggested. In vivo animal models complete the testing battery. Some issues, such as in vivo relevance, gender difference, age and ontogeny issues can only be addressed using in vivo models. Transporter specificity is provided by using knock-out or mutant models. Alternatively, chemical knock-outs can be employed. Compensatory changes are less likely when using chemical knockouts. On the other hand, specific inhibitors for some uptake transporters are not available, limiting the options to genetic knock-outs.
