Translating PROTAC chemical series optimization into functional outcomes underlying BRD7 and BRD9 protein degradation

Proteolysis targeting chimeras (PROTACs) are complex molecules to design and optimize as degraders, primarily because the linker that bridges the two binding ligands is a highly variable element of design, yet critical for simultaneous engagement of target and E3 ligase into a ternary complex, target ubiquitination and ultimately degradation. These chemical and mechanistic complexities mean that the PROTAC design process can be a daunting endeavour, and it remains unpredictable how to best optimize them into high-quality degraders. To understand how medicinal chemistry decisions could translate into functional outcomes, here we present a retrospective and holistic mechanistic study of a two-part sequential medicinal chemistry program, termed Series 1 and 2, which previously led to the discovery of VZ185, a potent VHL-based dual BRD7 and BRD9 degrader. Analysis of the initial Series 1 compounds across three different cell lines containing endogenously tagged CRISPR HiBiT BRD7 or BRD9 revealed only partial degradation of BRD9, and no degradation of BRD7. Analysis of Series 2 PROTACs, which was designed based on the degradation outcomes of Series 1 and in the absence of ternary complex structural information, showed the emergence of three lead compounds improved in BRD9 degradation and with additional specificity for BRD7. Biochemical analysis to interrogate ternary complex affinity and cooperativity demonstrated how subtle chemistry alterations impacted these parameters both positively and negatively, though on average, achieving only minor improvements in Series 2. In contrast, the greatest change between Series 1 and 2 was an improvement in cellular permeability, with the three lead degradation compounds showing high permeability. Lastly, cellular ubiquitination studies were performed and demonstrated the most potent degrader, VZ185, was the most robust for ubiquitination despite neither being the most permeable nor the best at forming ternary complex within the entire set. VZ185 and similarly active compounds were found to be efficacious degraders across all cell lines tested. Our mechanistic characterization provides insights that, while our structurally unguided medicinal chemistry campaign improved most notably cell permeability, increased degradation outcomes also required retaining productive ternary complex formation and target ubiquitination. •Mode-of-action screens offer insights into med-chem optimized BRD7/9 PROTACs.•Degradation screening in thr