The Investigation Of Rapid Drug Screening Method Using Enzymes Immobilized In Nanomaterials

Rapid screening of drugs at the molecular level is important step in drug discovery. It can be used in drug screening from hit drug to lead drug and other drugs. It has advantages on a clear role, less samples, etc. Enzyme is one important drug target, among the rapid drug screening methods, the free enzyme has the disadvantages of inactivation, poor stability and can not be reused, therefore, the method which has repeatability and stability is important to ensure the repeatability and stability of rapid drug screening. This investagation using liposomes and mesoporous materials respectively immobilized enzymes to establish drug rapid screening models, and using these models to screen drugs rapidly.This paper is divided into two parts:1. The method on liposome immobilized target enzymes drug rapid screeningUsing liposome immobilized carbon anhydrase B and liposome immobilized α-glucosidase to establish the rapid drug screening models of treatment of glaucoma and diabetes.The average size is 84.71 nm, stable liposomes were synthesized by film dispersion-ultrasonic method. The Zeta potential of liposomes is-48.56 mV. By using mobility shift method to ensure the interaction between enzyme and liposome. The results show that the binding constant between liposomes and carbonic anhydrase B was 9.306×102(g/m L)-1, and the binding constant between liposomes and α-glycosidase is 9.579×103(g/m L)-1. This result shows that both enzymes are capable of binding to the liposome.The particle size of liposome immobilized carbonic anhydrase B is larger than liposme, reached 99.43 nm. And its Zeta potential is-45.06 mV, a stable state. 4-carboxybenzenesulfonamid was used to establish the model of drug screening on glaucoma using carbonic anhydrase B as target. The binding constant betweent 4-carboxybenzenesulfonamid and liposome immobilized carbonic anhydrase B using Scatchard method is 1.172×104(g/mL)-1. Based on this model, 12 monomeric compounds including caffeic acid, 4-chloro-3-sulfamoylbenzoic acid, 2,4-dichloro-5-sulfamoylbenzoic acid and L-ascorbic acid were investigated. Among them, 4 monomeric compounds including caffeic acid and L-ascorbic acid have higher binding constant than 4-carboxybenzenesulfonamid and liposome immobilized carbonic anhydrase B, the interaction between them with liposome immobilized carbonic anhydrase B is strong.The particle size of liposome immobilized α-glycosidase is larger than liposme, reached 106.24 nm. And its Zeta potential is-48.56 mV, a stable state. Caffeic acid and gallic acid were used to establish the model of drug screening on diabetes using α-glycosidase as the target. The binding constants betweent caffeic acid or gallic acid and liposome immobilized α-glycosidase using Scatchard method are 1.186×104(g/mL)-1 or 9.579×103(g/m L)-1 respectively. Based on this model, 12 monomeric compounds including chlorogenic acid, riboflavin, protocatechuic acid, salicylic acid and ferulic acid were investigated. Among them, 5 monomeric compounds including chlorogenic acid and salicylic acid have higher binding constant than gallic acid and liposome immobilized α-glycosidase, the interaction between them with liposome immobilized α-glycosidase is strong.2. The method on mesoporous immobilized target enzymes drug rapid screeningA diabetes drug rapid screening model was established on mesoporous material immobilized α-glucosidase.9 mesoporous materials with different pore diameters and modifed groups were synthesed using 3 methods. Studying the effects of α-glucosidase immobilized in various materials to screen which is best materials to immobilize α-glucosidase. Mesoporous materials. The results showed that the-COOH-PMOs with 13.38 nm pore diameter is suitable to immobilize α-glucosidase, and the optimal immobilization conditions is pH4.5, incubation for 6~7 h. The effect of ethanol, methanol, isopropyl alcohol, acetone, acetonitrile on-COOH-PMOs immobilized α-glucosidase enzyme stability was studied. The results show that when 25% ethanol –NaAc buffer as mobile phase, the enzyme activity are higher 90%.Acarbose was used to estabilish the model of drug screening on diabetes using α-glycosidase as the target. When the concentration of acarbose was 0.2 mmol/L, the activity of immobilized enzyme is fully suppressed, after removing acarbose, the activity can be recovered, and the mesoporous immobilized α-glycosidase can be reused. Using sodium acetate buffer as the mobile phase, the interaction between α-glycosidase and 20 monomeric compounds including gallic acid, protocatechuic acid, ferulic acid and minoxidil were investigated. Among them, 7 monomeric compounds including gallic acid and minoxidil have strong inhibitory effect.20 traditional Chinese medicines(TCM) aqueous extracts including were prepared. Using sodium acetate buffer as the mobile phase, the interaction between α-glycosidase and 20 traditional Chinese medicines’ aqueous extracts were investigated. The result shows that there were 11 traditional Chinese medicines aqueous extracts which showed asignificant inhibitory effect, including Rhus chinensis and Spatholobus suberectus. 5 traditional Chinese medicines including Rhodiola rosea may have the ability of increasing the activity of α-glycosidase. The water-soluble of many compounds in natural product is poor, in order to study the interaction between water insoluble compounds and α-glucosidase action 15 TCM’s ethanol lysates were prepared. Using 25% ethanol-sodium acetate buffer as the mobile phase, the interaction between α-glycosidase and 15 TCM’s ethanol lysates were investigated. There were 5 Chinese medicines extracts which showed asignificant inhibitory effect, they are Rhus chinensis and Folium mori Fruit. And the extracts from 5 traditional Chinese medicines, including Rhodiola rosea and Ophiopogon japonicus may have the ability of increasing the activity of α-glycosidase significant activation.This study established drug rapid screening models using liposome immobilized carbonic anhydrase B, liposome immobilized α-glycosidase and mesoporous immobilized α-glycosidase. Based on these models, multiple drugs were screened, some of them inhibit the activity of the enzyme. So these models can be used in the drug screeing of glaucoma and diabetes.