Iterative Design and Optimization of Initially Inactive Proteolysis Targeting Chimeras (PROTACs) Identify VZ185 as a Potent, Fast, and Selective von Hippel–Lindau (VHL) Based Dual Degrader Probe of BRD9 and BRD7

Abstract

Developing PROTACs to redirect the ubiquitination activity of E3 ligases and potently degrade a target protein within cells can be a lengthy and unpredictable process, and it remains unclear whether any combination of E3 and target might be productive for degradation. We describe a probe-quality degrader for a ligase–target pair deemed unsuitable: the von Hippel–Lindau (VHL) and BRD9, a bromodomain-containing subunit of the SWI/SNF chromatin remodeling complex BAF. VHL-based degraders could be optimized from suboptimal compounds in two rounds by systematically varying conjugation patterns and linkers and monitoring cellular degradation activities, kinetic profiles, and ubiquitination, as well as ternary complex formation thermodynamics. The emerged structure–activity relationships guided the discovery of VZ185, a potent, fast, and selective degrader of BRD9 and of its close homolog BRD7. Our findings qualify a new chemical tool for BRD7/9 knockdown and provide a roadmap for PROTAC development against seemingly incompatible target–ligase combinations.

Extended pharmacodynamic responses observed upon PROTAC-mediated degradation of RIPK2

“…An appreciation of the significant opportunities afforded by recent developments in PROTAC-mediated targeted protein degradation for both novel therapeutics and tools for chemical biology is clearly exemplified by the dramatic increase in publications over the last two years [1][2][3] . Many of these reports have presented in vitro studies that have demonstrated the importance of the ternary complex between the target protein, the PROTAC, and the E3 ligase complex in determining not only the degradation efficiency, but also introducing greater degradation selectivity as compared to the native binding selectivity of the target binding moiety of the corresponding PROTAC [33][34][35][36] . In the current work, we began with a highly potent and selective RIPK2 inhibitors already available as a result of previous RIPK2 lead optimization efforts 25,37 , and we were able to quickly demonstrate effective degradation of the target protein with PROTACs based on these inhibitors.…”

Systematic exploration of different E3 ubiquitin ligases: an approach towards potent and selective CDK6 degraders

“…Despite the extraordinary importance of VHL in the PROTAC eld, this E3 ligase has not yet been utilized for the successful development of CDK4/6 PROTACs and was even considered to be inappropriate. 37 We followed a combinatorial approach towards VHL-based PROTACs by assembling four different linkers of various lengths and lipophilicities and two VHL ligands bearing different exit vectors for linker connection, 43,61,62 which were ultimately incorporated into the nal palbociclib-derived CDK4/6d. Noteworthy, all PROTACs of the rst, 'amide' subseries (Tables 2 and S6, ESI †) had pronounced ability to suppress CDK4/6 protein levels.…”

SPR-measured dissociation kinetics of PROTAC ternary complexes influence target degradation rate

“…Although functional degraders can be generated in the absence of positive cooperativity, 8,10 mounting evidence suggests enhancing cooperativity and stability of ternary complexes could be an effective strategy in PROTAC design. [11][12][13] As bifunctional molecules, PROTACs are subject to a well-recognised 'hook effect', whereby at high PROTAC concentrations, binary interactions may outcompete ternary complex formation. 5,143 Cooperativity is expected to counter this 'hook effect' often exhibited by bifunctional molecules, thereby widening the concentration window for PROTAC activity, and could also enable use of weaker-affinity ligands.…”

“…fluorescence polarization, to measure PROTAC binding to VHL in the presence or absence of target protein) have also been used to determine binding and cooperativity of PROTACs. 11 ITC has also been used for label-free direct quantification of thermodynamic and binding parameters of PROTACs (for both binary and ternary complex formation), 2, 10, 12 however is relatively more resource intensive and lower throughput. Notably, none of these assays offer kinetic resolution.…”