Small Molecular β-secretase Inhibitor Discovery And Glucokinase Activator Evaluation Correlating With Related Mechanisms Analysis

With the increase of the aging population, the incidence of dementia has become larger. Alzheimer's disease (AD) is the most common form of the irreversible dementia among the old people. The major hallmark of AD at the histopathological level is the accumulation ofβ-amyloid peptide (Aβ) in brain. Acting as a rate-limiting enzyme,β-secretase plays important roles for Aβformation. Therefore,β-secretase has thus been putatively considered as a promising drug target for the treatment and prevention of AD. In this work, with related molecular and cellular techniques, we constructed theβ-secretase expression plasmid and set up a high-efficiencyβ-secretase expression system against TN cells. Based on the fluorescence resonance energy transfer (FRET) technology, we established an HTS platform in the screening and evaluation ofβ-secretase small molecular inhibitor. By screening over 700 compounds against the lab-constructed natural product library, it is discovered that 2 natural products (NPLC362,NPLC364) showed strongβ-secretase inhibition activities. With these two acitive compounds as structural models, we also synthesized 10 related analogs whoseβ-secretase inhibition activities were also evaluated. The preliminary animal model based assay indicated that compound NPLC362 could improve the learning ability and mend the Aβ1-40 caused memory impairment for the tested Alzheimer's rats, implying that NPLC362 might be used as a lead compound for anti-AD drug design.Hyperglycemia caused by disorder of glucose homeostasis is one of the characteristics of type-II diabetes. The occurrence of type-II diabetes correlates with combined defects in both insulin secretion and insulin action. Glucokinase (GK) plays preeminent roles in glucose sensor and glucose homeostasis, the activation of GK enzymatic activity thus could show important clinical benefits for the treatment of type-II diabetes. In this work, we constructed a human liver glucokinase expression plasmid based on corresponding molecular biology theories and bioengineering techniques, and set up a high-efficiency expression system in E.coli. With surface plasmon resonance (SPR) technology, we established a high through-put (HTS) system for GK small molecular binding affinity screening and evaluation. Based on GK activity assay, we further constituted a GK small molecular activator evaluation system. Through the screening against the lab compound library, we discovered 43 compounds with strong GK binding affinity and 2 compouds with GK activation activities. To make rational structural optimization and design for GK compounds, we also carried out related study of the GK substrate-catalyzing mechanisms on the basis of computer molecular dynamics simulations. The mutation results of 7 single-amino-acid and 2 double-amino-acid mutants revealed that lysine 169 (K169) was a critical amino acid for glucose phosphorylation catalyzed by GK. K169 mutation could cause the total inactivation of GK. The double mutant in lysine 56 (K56) and glutamic acid 256 (E256) sites totally inactivates GK. Based on the relevant computational analysis, we suggested that the double mutant disrupted the whole ATP-binding domain formed by K56 and E256, causing GK inactivation. The revelation of the key amino acids information of GK would be a benefit to GK activator design and structural optimization targeting this kinase.