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Development of a fast CE method for high throughput screening of ecto-5'-nucleotidase inhibitors
Rabia Raza1, Yu Bai1, Huwei Liu1
1Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Institute of Analytical Chemistry, College of Chemistry and Molecular Engineering, Peking University, Beijing, P. R. China.
Abstract:
Overexpression of ecto-5'-nucleotidase is found to be linked to cancer progression and other diseases which makes screening of its inhibitors on demand. Unfortunately, there is scarcity of reported ecto-5'-nucleotidase inhibitors because of unavailability of simple, fast and efficient methods to study its inhibition. In this study, we have developed a very fast, reverse polarity capillary electrophoresis based method for screening of ecto-5'-nucleotidase inhibitors. Both EOF and electrophoretic mobility were maintained in same direction by dynamically coating lining of capillary and applying negative voltage, resulting in appearance of both adenosine monophosphate (AMP) and adenosine peaks in less than 2 min. Separations were performed in reverse polarity using -20 kV. Hexadimethrine bromide (HDB) was used as a buffer additive (0.025% in 40 mM borate buffer). An online sample stacking was attained by a special sweeping effect which enabled a long (65 s) injection time without compromise in peak shape. Using this newly developed method, both AMP and adenosine peaks were obtained with a baseline resolution, in less than two minutes. Method was validated by measuring km , and Vmax values of human ecto-5'-nucleotidas and Ki value of standard inhibitor quercitin against this enzyme. Method is simple and fast and can be conveniently optimized for high throughput screening of ecto-5'-nucleotidase inhibitors.
Insights
Screening for ecto-5'-nucleotidase inhibitors is crucial for disease treatment. A new, rapid capillary electrophoresis method allows for fast and efficient screening of these inhibitors, aiding drug discovery.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Pharmacology
Background:
- Ecto-5 -nucleotidase overexpression is linked to cancer and other diseases.
- A lack of efficient screening methods hinders the discovery of ecto-5 -nucleotidase inhibitors.
Purpose of the Study:
- To develop a fast, efficient capillary electrophoresis method for screening ecto-5 -nucleotidase inhibitors.
- To enable high-throughput screening of potential ecto-5 -nucleotidase inhibitors.
Main Methods:
- Developed a reverse polarity capillary electrophoresis method.
- Utilized dynamic capillary coating and negative voltage for separation.
- Employed Hexadimethrine bromide as a buffer additive.
- Incorporated online sample stacking with a sweeping effect for enhanced sensitivity.
Main Results:
- Achieved baseline resolution of adenosine monophosphate (AMP) and adenosine peaks in under 2 minutes.
- Demonstrated method validation by determining kinetic parameters (Km, Vmax) and inhibitor constants (Ki).
- Confirmed the method's simplicity, speed, and suitability for high-throughput screening.
Conclusions:
- The developed capillary electrophoresis method is a significant advancement for ecto-5 -nucleotidase inhibitor screening.
- This method facilitates faster identification of potential therapeutic agents for diseases associated with ecto-5 -nucleotidase.
- The technique's efficiency supports drug discovery and development efforts.