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Targeted protein degradation (TPD) of neo-substrates with proteolysis targeting chimeras (PROTACs) or molecular glues has emerged as a key modality in exploring new biology as well as designing new drug candidates where catalytic inhibition is neither efficacious nor an option. TPD is mediated through harnessing E3 ligases and redirecting them to ubiquitinatede novotarget proteins for subsequent proteasomal degradation. Until recently, E3 ligase chemical matter available for mediating TPD has been limited to a relatively low number of ligases, considering that over 600 E3 ligases are encoded by the human genome. In addition, the most utilized ligase for TPD approaches, CRBN, has been observed to be downregulated in settings of acquired resistance to immunomodulatory inhibitory drugs (IMiDs). IMiDs are molecular glues that target IKZF transcription factors to CRBN for degradation. Resistance is potentially accelerated by non-essentiality of CRBN for cell viability. Here we investigated if the essential E3 ligase receptor DCAF1 can be harnessed for TPD utilizing a potent, non-covalent DCAF1 binder. We show that this binder, selective for the CRL4DCAF1E3 ligase complex, can be functionalized into an efficient DCAF1-BRD9 PROTAC. Chemical and genetic rescue experiments confirm specific degradation via the CRL4DCAF1E3 ligase. We further highlight the versatility of DCAF1 for TPD by developing a DCAF1-dasatininb PROTAC targeting multiple cytosolic and membrane bound tyrosine kinases. We expand these findings towards Bruton’s tyrosine kinase (BTK) selective PROTACs and through extensive optimization and characterization efforts share key observations that led to a potent and selective DCAF1-BTK PROTAC (DBt-10). Finally, with this PROTAC DBt-10, we show rescue of BTK degradation in a BTK-dependent, CRBN-degradation-resistant cell line and provide a rationale for E3 ligase swap to overcome CRBN mediated resistance.

Propofol, a dialkylphenol intravenous anesthetic, has been widely administered for over a quarter century in general anesthesia and for rapid sedation. Regardless, the drug's mechanism of action remains elusive with mounting evidence suggesting contributions from multiple pathways. Most proposed targets, such as ion channels and ligand gated receptors, are low abundance but functionally important proteins. To uncover these significant targets, we synthesized a photoactive- and ‘clickable’- propofol analogue with meta-trifluoromethyldiazirinyl and ortho-(propynyloxy)methyl substitutions in place of the two isopropyl groups. This novel o-alkynyl-m-azipropofol maintains the physiochemical, biochemical and in vivo properties of propofol. It also photoadducts to known propofol binding sites in model proteins. Once adducted, the alkyne click moiety successfully undergoes copper catalyzed 1,3-dipolar cycloaddition reactions with azide-rodamine and -biotin tags allowing for fluorescent labeling and avidin resin enrichment of photolabeled targets within ex vivo systems. Using fluorescent microscopy or mass spectroscopy, the chemically ‘dual-active’ propofol analogue allows localization and identification of key targets in the biochemical pathways that underlie alkylphenol induced anesthesia.