Functional Study Of ?-Glucosidase Inhibitors

Type ? diabetes?non-insulin-dependent diabetes mellitus?is an endocrine and metabolic disease caused by defective islet ?-cell function or insulin resistance,and is characterized by long-term hyperglycemia.?-Glucosidase?EC 3.2.1.20?is a carbohydrate hydrolase,and the inhibition of its activity may delay the digestion of carbohydrates and thereby reduce the amount of glucose absorbed into the blood.Thus,?-glucosidase is considered to be an important target for the treatment of noninsulin-dependent diabetes mellitus.Several incompletely satisfactory therapeutic drugs are already on the market due to the high cost and toxic side effects.Therefore,designing novel,safe and effective?-glucosidase inhibitors is an attractive goal in the field of medicinal chemistry.Polyoxometalates?abbreviated as POMs?,due to their incomparable structural diversity,novel functional properties and lower cytotoxicities,have shown good application prospects in the field of medicinal chemistry,especially of the Keggin and Dawson-type with superior inhibitory properties and lower cytotoxicities may result in potentially effective enzyme inhibitors.In addition,Spirulina species are edible with high nutritional as well as potential therapeutic values.However,so far no research has reported the use of purified phenolic extracts isolated from Spirulina,and phenolic acids have the potential of anti-hyperglycemia,so Spirulina phenolic acids has potential value in the development of functional foods and therapeutic drugs for type ? diabetes.Herein,the inhibitory effects and mechanisms of Spirulina phenolic acids and POMs affected by three crucial factors?heteroatom,transition metal substitution element and vanadium substitution number?on?-glucosidase were studied and the corresponding inhibition kinetics and molecular simulation performed.Main contents and results from these experiments are as follows:?1?Three Keggin-type POMs(H₅GaMo₁₂O₄₀,H₄SiMo₁₂O₄₀0 and H₃PMo₁₂O₄₀)with different types of heteroatoms?central atoms?were synthesized and characterized,and corresponding inhibition kinetics and molecular simulation were performed.Our kinetic data showed that H₃PMo₁₂O₄₀(IC₅₀=6.14±0.38?M)has the strongest inhibitory effect on?-glucosidase,and its inhibitory effect was approximately 117 times higher than that of standard acarbose,and all synthetic compounds exhibited reversible competitive inhibition.Moreover,the structure and composition of POMs may change their own central atomic charge charge,oxidation-reduction and acidity,which may significant influence the inhibition behaviour of POMs on?-glucosidase.According to the molecular simulation,all compounds we synthesized competitively bind to the active site of?-glucosidase mainly through multiple van-der-Waals and hydrogen bond interactions.?2?Different transition metal-substituted Seven Keggin-type POMs Na_9-n-n PMo₁₁MⁿO₄₀?Mⁿ=Ni^?,Mn^?,V^?,Zn^?,Co^?,Cr^?,Fe^??were synthesized and characterized,and corresponding inhibition kinetics and molecular simulation were performed.Our kinetic data showed that PMo₁₁Ni(IC₅₀=37.29±1.72?M)has the strongest inhibitory effect on?-glucosidase,and its inhibitory effect was approximately 20 times higher than that of standard acarbose.Combining the inhibition mechanism and the type of inhibition,we found that all synthetic compounds except PMo₁₁Ni?reversible mixed-type?and PMo₁₁Fe?reversible noncompetitive?exhibited reversible competitive inhibition.In addition,the Inhibitory type,oxidation-reduction and transition metal substitution element of POMs may also indirectly influence the inhibition behaviour on?-glucosidase.Molecular docking simulation suggested that PMo₁₁Ni forms five hydrogen-bond interactions with the amino acid residues?Ser240,His280,Ser311,Pro312 and Arg315?and forms van der Waals interactions with the polar amino acid?Tyr158,Gln279,Asp307 and Thr310?around the active region.?3?Vanadium-substituted five Dawson-type POMs H_6+nP₂Mo_18-nV_nO₆₂?n=1-5?were synthesized and characterized,and corresponding inhibition kinetics and molecular simulation were performed.Our kinetic data showed that P₂Mo₁₅V₃(IC₅₀=57.01±2.11?M)has the strongest inhibitory effect on?-glucosidase,and its inhibitory effect was approximately 13 times higher than that of standard acarbose,and all synthetic compounds exhibited reversible competitive inhibition.Moreover,the increase in the number of vanadium substitutions?not more than 3?will directly affect its acid strength and oxidizing properties,and then may indirectly influence its binding affinities to the active site of the?-glucosidase.Therefore,the more the V-containing component?not more than three?,the stronger the inhibitory effect on?-glucosidase.Molecular docking simulation suggested that P₂Mo₁₅V₃ forms six hydrogen-bond interactions with the amino acid residues?Tyr158,Asp242,His280 and Ser311?and forms van der Waals interactions with the polar amino acid?Ser240 and Arg315?around the active region.?4?Spirulina phenolic acids were extracted with methanol and identified as4-hydroxycinnamic acid,and its inhibition kinetics and mechanism of?-glucosidase were studied.Our kinetic data showed that 4-hydroxycinnamic acid(IC₅₀=1.67±0.02 mM)has a potential inhibitory effect on?-glucosidase and exhibited reversible mixed-type inhibition.Molecular docking simulation suggested that?-glucosidase inhibitory activity depends on the phenolic hydroxyl and carboxyl groups of 4-hydroxycinnamic acid,which forms hydrogen bonds with enzyme active sites amino acid?Gln279 and Arg315?to promote steric hindrance,conformational changes,and suppression of enzymatic activity.Moreover,4-hydroxycinnamic acid also forms van-der-Waals forces with the polar amino acids?Tyr158,Tyr 316,Asp352,Gln353,Glu411 and Arg442?around the active region.These polar active-site residues significantly contribute to the binding of the 4-hydroxycinnamic acid.