Rapid screening of enzyme inhibitors using capillary electrophoresis with enzyme-based biosensors.

摘要

Drug design based on inhibitor/enzyme interaction is a common approach in the pharmaceutical industry. An analytical tool for identifying enzyme inhibitors would be useful in the drug development process. The system described here employed capillary electrophoresis as the high performance separation technique, coupled with on-line electrochemical detection of biological activity using a microscale enzyme-based biosensor. Several enzyme biosensor systems were examined, including acetylcholinesterase, {dollar}alpha{dollar}-chymotrypsin and pancreatic elastase. The primary focus was on the use of acetylcholinesterase-based biosensors. Acetylcholine was added to the run buffer and hydrogen peroxide, the electroactive product of the enzyme reaction, was oxidized at the surface of the electrode. In this way, the enzyme activity was continuously monitored. When a separated zone containing an inhibitor interacted with the sensor, the enzyme activity was decreased with a corresponding reduction in substrate turnover. This decrease in substrate production was the measured analytical response indicating the presence of an inhibitor. The construction and characterization of these biosensors, in addition to their combination with capillary zone electrophoresis, is described. The response of the system to several pharmaceutically relevant inhibitors such as neostigmine bromide and 2-PAM that were separated by capillary zone electrophoresis was demonstrated. The response profile of the sensor could be used to differentiate between reversible and irreversible inhibitors and the response of the sensor to inhibitors was concentration dependent. E. Green Mamba snake venom, which contained the potent AChE inhibitor fasciculin was used as an example of how this system could identify enzyme inhibitors even in complex biological samples. A technique in which the identified inhibitors were collected onto a membrane at the end of the capillary for analysis by off-line MALDI-TOF mass-spectrometry was described. The adaptation of these biosensors for use in a format suitable for high throughput screening was also introduced. In this format the biosensors were used singly or in a small array to screen samples in microtiter plates. This may be useful for screening combinatorial libraries where the target species is an enzyme inhibitor.