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Published on: August 17, 2019
Hexokinase inhibitor screening based on adenosine 5'-diphosphate determination by electrophoretically mediated
1Shanghai Institute of Organic Chemistry, Chinese Academy of Science, Shanghai, PR China.
Electrophoresis
|March 24, 2009
Summary
A new capillary electrophoresis method (EMMA) efficiently screens hexokinase inhibitors by measuring adenosine 5'-diphosphate (ADP) production. This simple, cost-effective technique offers robust and high-quality screening data for drug discovery.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Chemical Biology
Background:
- Hexokinase is a crucial enzyme in glucose metabolism.
- Developing efficient screening methods for hexokinase inhibitors is important for therapeutic research.
- Existing methods may lack speed, cost-effectiveness, or robustness.
Purpose of the Study:
- To develop a capillary electrophoresis (CE) based method for high-throughput screening of hexokinase inhibitors.
- To optimize reaction and separation conditions for accurate measurement of enzyme activity.
- To validate the method's performance for inhibitor screening.
Main Methods:
- Electrophoretically mediated microanalysis (EMMA) combining on-column reaction and detection.
- Assay of hexokinase activity by measuring adenosine 5 -diphosphate (ADP) production using UV detection.
- Optimization of buffer compositions and capillary coating for improved separation and reduced analysis time.
Main Results:
- Optimized conditions yielded effective on-column enzymatic reaction and separation of adenosine 5 -triphosphate (ATP) and ADP.
- Dynamic capillary coating with hexadimethrine bromide (HDB) reduced separation time to under 3 minutes and minimized peak tailing.
- Achieved a Z' factor of 0.98, indicating high-quality screening data.
Conclusions:
- The developed EMMA method is a simple, robust, and cost-effective approach for hexokinase inhibitor screening.
- This method provides a reliable platform for identifying potential therapeutic agents targeting hexokinase.
- The optimized CE technique offers significant advantages in speed and data quality for biochemical assays.
