A Gold-PROTAC degrades the oncogenic tyrosine kinase MERKT: insights into the degradome from a steady-state system

Targeted protein degradation is a valuable strategy to eliminate disease-relevant proteins. Among other classes, proteolysis targeting chimeras (PROTACs) are bifunctional molecules that induce the proximity of a recruiter E3 ligase and the target protein of interest for ubiquitination and subsequent degradation by the proteasome. While most PROTACs are non-covalent interactors, covalent PROTACs may benefit from increased selectivity and improved pharmacodynamics, yet remain largely understudied. In this context, a gold-based PROTAC (AuPROTAC) was synthesized, consisting of a cyclometalated Au(III) warhead, known to induce covalent cysteine-arylation in a gold-templated two-step mechanism, linked to a cereblon binding moiety. The degradome of the covalent AuPROTAC was characterized by establishing a cycloheximide chase assay, which was performed in a non-proliferative steady-state HL-60 cell culture system that maintained a static protein turnover and efficiently decoupled protein degradation from down-regulation. The method was verified with the known SMARCA2 and PBRM1-degrader ACBI2. Interestingly, amongst the possible targets, AuPROTAC could degrade the oncogenic tyrosine kinase MERTK and the thioredoxin-like 1 protein TXNL1, while their degradation was successfully rescued by proteasome inhibition. The investigated PROTACs were found to affect protein half-lives; therefore, proteome-wide degradation selectivity was further characterized by ranking degraded targets based on the acceleration of protein half-lives. Interestingly, the AuPROTAC degraded a relatively limited number of proteins (95) when compared to the targeted ACBI2 (221). Overall, the established approach can be used to efficiently explore degradomes of CRBN and VHL recruiter-based PROTACs.