Novel Approaches for PROTAC Drug Discovery

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PROteolysis TArgeting Chimeras (PROTACs) is a new therapeutic class comprised of small molecules binding a target protein and a ubiquitin (Ub) E3 ligase, enabling selective target degradation. PROTACs’ advantages include exquisite selectivity, tolerance of weak binders, and maximal degradation with limited target engagement. The first PROTACs employed the E3 ligase component pVHL to degrade target proteins. Around the same time, thalidomide and related analogs (IMiDs) were successfully repurposed as anti-cancer agents, and subsequently their ubiquitin ligase-associated molecular mechanism was discovered, when thalidomide was shown to bind to cereblon (CRBN), a pVHL-like subunit of a Cullin 4-type ubiquitin ligase. IMiDs promote interaction of this E3 ligase transcription factors that control T cell immunity. In cells, Ub-mediated signaling regulates protein content, location, and activity, primarily through protein degradation, and dysregulation of ubiquitin ligases is linked to numerous devastating diseases. Thus, ligases are promising drug targets and vehicles for PROTACs. Of ~700 Ub ligases, only CRBN and pVHL have been exploited for PROTAC development, a process hindered by several issues. There is a disconnect between the rapid synthesis of new PROTACs by chemists and the time- consuming, artifact-susceptible immunoblot cell assays used to evaluate them. Moreover, binding of a PROTAC to its target does not ensure degradation, owing to steric hindrance, ubiquitylation at the wrong site or in the wrong chain configuration (K63 vs K48), or metabolism inside or poor penetrance into cells. Thus, it is difficult for PROTAC chemists to develop meaningful structure activity relationships, which are essential for preclinical development. It is proposed here to develop ligase-selective, high-throughput cellular assays for PROTAC- mediated ubiquitylation of target proteins. In phase I, LifeSensors will employ unique affinity matrices called TUBEs (Tandem Ubiquitin Binding Entities) in a high throughput mode to analyze ubiquitylation patterns of endogenous proteins in cells. This approach offers the potential to expedite discovery of novel PROTACs, establish a relationship between ubiquitylation and degradation, eliminate low throughput, time-consuming western blot analysis, and lead to the timely identification and development of novel PROTAC drugs, as medicinal chemists will be able to design PROTACs efficiently and rationally, eventually encompassing both degradative and non-degradative ubiquitylation. Phase I will be accomplished by establishing a clear relationship between PROTAC-mediated ubiquitylation and degradation for CRBN and HDM2 ligase target proteins in cells and adapting LifeSensors’s TUBEs technology to monitor PROTAC ubiquitylation and degradation in a high throughput fashion in cells. In Phase II the technology will be expanded to entire ligase families.