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05:33 min
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November 9th, 2020
DOI :
November 9th, 2020
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Introduction
0:50
Endpoint Degradation Studies with HiBiT CRISPR Target Proteins in Lytic Format with Optional Cell Viability Fluorescence Analysis
3:07
Results: Degradation Compound Analysis in a HiBiT CRISPR Cell Line
4:51
Conclusion
副本
This approach allows for cellular monitoring of protein degradation and rapid screening of degrader compounds. The dynamics of degradation can be monitored in a cellular context in a highly sensitive and quantitative manner, allowing for accurate determination of key degradation parameters. These methods allow for HTS profiling of degrader compound activity, enabling triaging in the early therapeutic discovery phase.
They also provide insight for anyone who wants to study modulation of endogenous target levels, whether increasing or decreasing. Demonstrating the procedure will be Celia Bisbach, a research scientist in my laboratory. Begin by preparing and plating mammalian adherent or suspension cells.
Adjust the cell density to 2.22 times 10 to the 5th cells per milliliter and dispense them into plates with a minimum of three wells per experimental and control condition. Prepare serially-diluted PROTAC, or degrader test compound plates, at 1000x concentration in 100%DMSO. Then dilute it to 10x final concentration in the cell culture medium.
Add an equal volume of DMSO to the medium to create a no-compound DMSO control. Add the appropriate amount of 10x compound or control solution to the cells in the plate and incubate the plates at 37 degrees Celsius in 5%carbon dioxide. To measure endpoint luminescent signal, prepare 2x lytic detection reagent by adding 20 microliters of lytic substrate and 10 microliters of LgBiT protein for every one milliliter of the lytic buffer.
Prepare enough reagent for all wells to be assayed, including extra volume to account for pipetting error. Add the prepared lytic detection reagent to cells and mix the plate on a microplate vortex mixer for 10 to 20 minutes at 350 RPM. Measure luminescence on luminometer capable of reading a 96 or 384 well plate.
If using cell viability multiplexing, do so before addition of the lytic reagent. 30 to 40 minutes prior to the desired endpoint measurement, prepare a 6x cell viability detection reagent solution by adding 10 microliters of the substrate to two milliliters of the assay buffer. Add the prepared reagent to wells and mix briefly on a microplate vortex mixer.
Then incubate the plate for 30 minutes in a 37 degree Celsius incubator. At the desired endpoint, measure fluorescence on an instrument capable of reading fluorescence in 96 or 384 well format. Prepare 2x lytic reagent as previously described.
Then add the reagent to the wells and mix the plate on a microplate vortex mixer for 10 to 20 minutes. After mixing, use a luminometer to measure luminescence. To demonstrate single concentration endpoint lytic degradation analysis, several CDK target proteins were endogenously tagged with HiBiT and treated with the pan kinase cereblon-based PROTAC TL12-186.
The CDK protein level was measured at different time points, and the fractional RLU was determined. To understand how CDK proteins directly compared to each other in terms of protein loss, the fractional RLUs were calculated as total percent degradation. A kinetic degradation analysis was performed by endogenously tagging BET family member proteins with HiBiT and HEK293 cells stably expressing the LgBiT protein.
The cells were then treated with three different concentrations of the pan-BET PROTACs. The cereblon-based dBET6, and the VHL-based ARV-771. Kinetic dose response degradation profiles of Ikaros IKZF1-HiBiT CRISPR Jurkat cells stably expressing LgBiT protein treated with four different molecular glue compounds are shown here.
Significant differences in degradation response amongst the compounds, as well as across the concentration series are observed. To quantitatively assess degradation and rank order of the compounds, the dose response profiles were used to calculate key degradation parameters including the degradation rate, degradation maximum, and Dmax50 values. Cell viability multiplex assays showed no loss in cell viability for the concentrations tested.
The most important steps in this protocol are determining the fractional RLU or percent degradation by normalization to the DMSO control. Following this protocol, one could perform a phenotypic assay to understand how loss of a particular protein can impact cellular health or function. In addition, these HiBiT cell lines can also be used to study protein interactions or small molecule binding using NanoBRET technology.
This protocol describes the quantitative luminescent detection of protein degradation kinetics in living cells that have been engineered using CRISPR/Cas9 to express antibody free endogenous protein detection tag fused to a target protein. Detailed instructions for calculating and obtaining quantitative degradation parameters, rate, Dmax, DC50, and Dmax50 are included.
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