Thread by @Chemist_Payne

On @biorxivpreprint we report a direct TR-FRET-based protein quantification strategy (<1 h from lysis➡️read) in unmodified cell lines. If you're into PROTACs, and you hate running Westerns, check it out! Mini-thread: (1/n)
biorxiv.org/content/10.110…

Two birds with one stone: using clever biochemistry principles and a single POI-targeting antibody, we can rapidly & quantitatively measure both endogenous target engagement and protein levels in whole cell extracts, here exemplified for BRD4 (2/n)

A conventional blood-sweat-and-tears Western blot takes the average scientist 2 days. With our strategy, we initially optimized the assay in a 24-well plate format. The readout takes <1 h. Comparing BRD4 levels measured by our platform to Western blot were nearly identical (3/n)

Often, PROTAC development demands that many variables (dose, time, degradation rescue) be analyzed systematically, which can quickly overwhelm the capacity of conventional Western analyses. In contrast, our assay can be run directly in 96-well plates, enabling HTS efforts (4/n)

We used our new platform to characterize celastrol, a p-quinone methide-containing triterpenoid, as a powerful E3 ligase recruiter for PROTAC development. Many of the >600 E3 ligases in the human proteome possess solvent-exposed Cys residues that celastrol can exploit (5/n)

Importantly, our approach reported here is target-agnostic and can readily be applied to any system of interest. All you need is a good antibody and a linker-modified POI-targeting ligand, which for PROTAC development has often been identified early on (6/n)

We look forward to how our approach will enable high-throughput screening efforts in the identification and characterization of new degraders! @mazit @TannousLab (7/n)
#TRFRET #PROTAC #PROTACs #degrader #chemicalbiology #smallmolecules