Establishing The Discovery And Evaluation System For Development Of α-glucosidase Inhibitors From Natural Products

Type2diabetes mellitus, is a metabolic syndrome that is characterized by continuous high level of glucose in the blood with defects in insulin secretion, insulin action or both. Controlling blood glucose levels is critical. One efficient strategy is to slow down the digestion process of ingested carbohydrates and starches and thus delay the glucose absorption by inhibiting the enzymes involved in the hydrolysis of dietary starches into glucose. Three a-glucosidases are involved in the complete digestion of dietary starches and sugars into glucose in intestine. The digestion is initiated by human pancreatic a-amylase (HPA) that hydrolyzing starch into shorter linear and branched dextrin chains. The resultant oligoglucans is eventually hydrolyzed at the nonreducing into glucose by two small-intestinal brush-border exohydrolases:maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI). MGAM has two independent functional catalytic domains. Therefore, a-glucosidase is becoming one of the crucial targets for anti-diabetes drugs research and development.Natural products have long proved to be valuable sources, and various enzyme inhibitors are derived from natural products. Screening a-glucosidase inhibitors for anti-diabetes drugs development from natural products has been a central topic in drug discovery. Firstly, a library of TCM extracts that including411kinds of TCM was constructed. A high-throughput screening (HTS) method was established for a-glucosidase inhibitors screening against MGAM-N, MGAM-C and HP A.23kinds of TCM extracts has significant MGAM-N inhibitory activity;20kinds of TCM showed notably inhibitory effect against MGAM-C;16kinds of TCM could inhibit the activity of HPA efficiently. Compared the TCM with a-glucosidase inhibitory activity, the TCM that has inhibitory activity against MGAM-N’and MGAM-C were different from the TCM with HPA inhibitory activity.20strains with MGAM-C inhibitory activity were found in the process of the high throughput screening of actinomycetes broth against MGAM-C. Pre-column derivatization HPLC method was applied for screening1-deoxynojirimycin (DNJ) producing strains. DNJ was detected from the broth of strain PW409. Polyphasic taxonomy studies verified strain PW409belonged to Streptomyces gobitrici. Biological activity based HPLC-Q/TOF method was established for screening and identifying a-glucosidase inhibitors in the broth of strain PW409, DNJ and miglitol was identified as the active compounds. The quantification analysis indicated the content of DNJ and miglitol were11.2mg/L and95.8mg/L. The studies provided new strain resource for α-glucosidase development.AIB656was extracted from the broth of Strain ZG656, which has extremely strong α-amylase inhibitory activity. The efficient MGAM-N and MGAM-C inhibitory activity were also comfirmed. Multi-targets based biological activity discovery and evaluation system was established for screening and identification of α-glucosidase inhibitors.51acarviostatins was identified in AIB656by UPLC-Q/TOF.5fractions had efficient MGAM-N inhibitory activity;8fractions showed strong MGAM-C inhibitory effect;9fractions demonstrated excellent HPA inhibitory activity. The biological practical screening and molecular docking results elucidated the smaller molecules showed remarkable MGAM-N inhibitory activity; Acarviostatin with3-5mimic sugar rings showed strong inhibitory ability against MGAM-C; The larger acarviostatins molecules (6-15mimic sugar rings) had significant HPA inhibitory activity. The results clarified the selectivities of the three different α-glucsosidase to various sizes inhibitors were quite distinct. Molecular docking and interaction study showed acarviostatin10-1firmly binded to the active site of MGAM-N by forming hydrogen bond with Asp327, Asp542, His600and Asp203. acarviostatin10-1occupied the binding site (subsite-1and+1) in the catalytic center of MGAM-N perfectly. Acarviostatin101spanned the binding site (subsite-1and+1,+2,+3) in the catalytic center of MGAM-C completely and formed stable binding state. The results clarified the mechanism in structure biology that acarviostatin10-1is the best MGAM-N inhibitors and acarviostatin101is the best MGAM-C inhibitors in acarviostatins.The pharmacokinetics and bioavailability of a-glucosidase inhibitor were studied. The result indicated that the absorption rate of a-glucosidase inhibitor was very fast. The Cmax of DNJ was0.8h, and the absolute bioavailability of DNJ was55.02%. Considering quickly absorption of DNJ would decrease the inhibitory activity in intestine, adjuvants was selected for changing the absorption characteristics. The pharmacokinetics of DNJ indicated the Cmax and AUC of DNJ were significantly decreased by CMCNa. CMCNa could postpone the absorption rate of DNJ in intestine. The oral glucose test verified the pharmacodynamics of DNJ was improved by CMCNa.The systematically a-glucosidase inhibitor discovery and screening approaches were established based the combination of HPLC-MS, molecular docking and enzyme evaluation. Comprehensive multi-targets including MGAM-N, MGAM-C and HPA based a-glucosidase inhibitor screening were proceeded against the extracts of TCM and secondary metabolites of actinomycetes. The reaults and structure biology studies clarified the distinct selectivities of the three a-glucosidases to various sizes inhibitors. The combination of different a-glucosidase inhibitors or composite intervention was probably more effective for diabetes treatment, which was of importance for the a-glucosidase inhibitors type anti-diabetes drugs research and development.