Establishment of hypoglycemic agent screening method based on human glucokinase.
OBJECTIVE
To establish a reliable platform for screening glucokinase activators (GKAs) in vitro.


METHODS
Pancreatic glucokinase (PGK) protein expressed in a prokaryotic expression system as a histidine-tagged fusion protein from Homo sapiens was produced. Then, response surface methodology (RSM) was used to optimize the microplate-based GKA screening platform. In the first step of optimization with Plackett-Burman design (PBD), initial pH, reaction time and MgCl2 were found to be important factors affecting the activity ratio of GKA (RO-28-1675) significantly. In the second step, a 2(3) full factorial central composite design (CCD) and RSM were applied to the optimal condition determination of each significant variable. A second-order polynomial was determined by a multiple regression analysis of the experimental data.


RESULTS
The following optimal values for the critical factors were obtained: initial pH 0 (7.0), reaction time-0.63 (13.7 min) and MgCl2 0.11 (2.11 mmol/L) with a predicted value of the maximum activity ratio of 34.1%.


CONCLUSIONS
Under the optimal conditions, the practical activity ratio is 34.8%. The determination coefficient (R2) is 0.9442, ensuring adequate credibility of the model. LLAE3, extracted from Folium nelumbinis in our laboratory, has prominently activated effects on PGK.
Wu
Chou-Fei
CF
Key Laboratory of State Food Science and Technology, Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang 330047, Jiangxi, China.
Xu
Yang
Y
Tao
Yong
Y
Yang
Ji-Yan
JY
D000704:Analysis of Variance; D055808:Drug Discovery / Q000379:methods*; D020536:Enzyme Activators / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D005941:Glucokinase / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007004:Hypoglycemic Agents / Q000032:analysis*; D007700:Kinetics; D013997:Time Factors
D020536:Enzyme Activators; D007004:Hypoglycemic Agents; D005941:Glucokinase