Oxford Drug Discovery Institute uses the FLIPR System to explore therapeutic mechanisms for neurodegenerative diseases
COMPANY/UNIVERSITY
Alzheimer’s Research UK funded Oxford Drug Discovery Institute, University of Oxford
TEAM MEMBERS
Dr. Franziska Guenther,
Dr. Katherine England,
Dr. James Taylor,
Dr. Emma Mead,
Prof. Paul Brennan,
Prof. John Davis,
Dr. Emma J. Murphy
PRODUCTS USED
FLIPR Tetra High-Throughput Cellular Screening System
ScreenWorks Software
The Challenge
Dr. Emma J. Murphy's research team is dedicated to exploring therapeutic approaches to neurodegenerative diseases. To do that, they study the effects of inhibitors on ubiquitin-specific protease 30 (USP30), which have shown potential as a therapeutic approach to promote mitophagy (the removal of damaged mitochondria) in neurodegenerative disorders.
To perform these calculations using high-throughput kinetic characterization, the team has developed a biochemical assay using the FLIPR® Tetra instrument. Prior to using the FLIPR® Tetra, the team used a third-party Microplate Reader to monitor the cleavage of a substrate called Ub-Rho by USP30. This method, based on measuring the increase in fluorescence, was excellent for determining IC50 values (the concentration of inhibitor that reduces enzyme activity by 50%) in a high-throughput manner. However, it had limitations in accessing early time points and making kinetic measurements. This was because the substrate had to be added to the plate before it was placed in the instrument, and the plate wells were read individually.
The Solution
Originally acquired for monitoring Calcium flux in cells, the FLIPR Tetra has proven to be an exceptional tool for investigating enzyme kinetics. In addition to Calcium Flux assays, the team now conducts various biochemical assays using the FLIPR Tetra, such as phosphate production assay and the aforementioned fluorogenic deubiquitinase assay.
The FLIPR Tetra has served as an invaluable platform for the team, enabling them to conduct high-throughput biochemical kinetic assays. Through this approach, they have been able to generate strong and reliable data while swiftly determining intricate kinetic parameters. The enhanced throughput achieved through this method enables the team to incorporate kinetic data into critical decision-making processes much earlier in the drug discovery journey.
Products Used
FLIPR Penta High-Throughput Cellular Screening System and ScreenWorks Software
The Results
A) The team has captured screenshots which show the increase of fluorescence overtime as the fluorogenic Ub-Rho substrate is cleaved by the enzyme under different inhibitor concentrations (Left). Additionally, they have also captured a screenshot of the entire plate, showcasing the impact of 16 different inhibitors in duplicate (Right).
To fully understand the therapeutic potential of USP30 inhibition as a mechanism for treating neurodegenerative diseases, it is necessary to conduct further efficacy studies using these inhibitors in relevant cellular and animal models. These investigations will shed light on the desired kinetic profile of USP30 inhibition and its effectiveness in combating neurodegenerative conditions.
References
Structural Premise of Selective Deubiquitinase USP30 Inhibition by Small-Molecule Benzosulfonamides