PROTAC (PROtein TArgeting Chimeric) is the ability to specifically degrade a protein using the endogenous machinery of the cell. This approach is applicable to elimination of established drug target proteins and the “undruggable” proteome.
PROTAC in drug discovery
PROTACs are highly appealing to drug developers as they have the potential to target the ‘undruggable’ proteome. Typically, the druggability of a protein depends on the inhibition of its activity. This is achieved by designing small molecules that can bind to a cavity or pocket in the protein of interest and block its function.
We generate a chimeric small molecule with two “warheads”, one that binds to the protein of interest, the second that binds to the E3 ligase and joined by a linker that brings both into close proximity. The ligase ubiquitinates the protein for degradation inside the cell.
How assays are developed for PROTAC
Charnwood Molecular has successfully employed screening strategies and methodologies for accurately measuring protein degradation.
WES and JESS (ProteinSimple) are used to monitor target protein levels in cells following treatment with PROTAC test compounds. It is highly quantitative compared to traditional Western blotting.
Proteins can be labelled either with Nanoluc or HiBiT (Promega) and luminescence can be used to monitor protein levels in living cells. Protein of interest is expressed with either Nanoluc or HiBiT as the label in cells (either transient or CRISPR).
Key factors for successfully quantifying protein degradation using these techniques are:
- Cell line with target expression at a relatively low level
- High quality primary antibody
- Loading control that is not overly-expressed and can be multiplexed with the target antibody
- Well-defined methodology for PROTAC drug treatment and sample preparation
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