Fig. 3. Screening platforms that will enhance TPD in the next decade. (A) Chemo proteomic competition experiments using promiscuous probes will uncover covalent binders of difficult to drug POIs and E3 ligases. Briefly, whole cells, cell lysate, or model organisms are treated with DMSO or the covalent ligand of interest. Proteins are then harvested and treated with a promiscuous probe outfitted with a terminal alkyne. Next, click chemistry is used to install a biotinylated light mass tag in the DMSO sample or a biotinylated heavy mass tag in the covalent ligand treated sample. The samples are then mixed in a 1 : 1 ratio, proteins are enriched by avidin pulldown, digested with trypsin, and finally biotin/avidin are removed via TEV cleavage. Purified proteins retaining the light and heavy tags are identified using mass spectrometry. Proteins with large light/heavy ratios are prospective targets of the covalent ligand (figure modified from Spradlin et al.⁹³). (B) DEL screening will be used to identify reversible ligands for POIs and E3 ligases. Epitope tagged protein can be purified from bacteria, insect, or human cells and then immobilized on affinity resin. Immobilized protein is then incubated with DNA barcoded libraries of millions to billions of compounds. Samples are washed and then bound molecules are eluted with mild heating. Eluted compounds are identified using next generation sequencing and can then be synthesized off DNA and tested in biophysical assays to assess binding.