PROTAC-based Methods as Traditional PROTAC Protein Degradation Technology Alternatives

In recent years, the transition from inhibition of aberrant protein function to specific degradation of desired proteins with Proteolysis Targeting Chimeras (PROTACs) has resulted in remarkable progress and is currently affecting a paradigm shift in drug discovery and therapy development. PROTAC approach has allowed quick expansion of the “druggable proteome” beyond proteins that bear distinct functional sites responsible for their respective mode of action. By avoiding high drug doses and acting via novel mechanisms, PROTACs show promise as therapeutic candidates for disease phenotypes that display resistance to conventional inhibitors.

Despite tremendous advancements outlined, current PROTAC approaches may still have undesired effects because the systemic application can affect untargeted tissue, a disadvantage shared with traditional inhibitor-based therapeutics. Therefore, alternative PROTAC methods capable of degrading these POIs have emerged, including RNA-PROTAC, oligonucleotide-based PROTAC, and TRAFTACs (transcription factor-targeting chimeras). In addition, new PROTAC formation models have been expanded, including light-controllable PROTACs (e.g., photoswitchable PROTACs, Photo-Cage PROTACs) and CLIPTACs (In-cell click-formed proteolysis targeting chimera).

Photoswitchable PROTACs

By merging the strategies of photopharmacology and small-molecule degraders, scientists introduce a novel concept for persistent spatiotemporal control of induced protein degradation that potentially prevents off-tissue toxicity. Building on the successful principle of bifunctional small-molecule PROTACs, they designed photoswitchable PROTACs (photoPROTACs) by including azobenzene linkers between both warhead ligands. PhotoPROTACs technique offers reversible on/off switching of protein degradation that’s compatible with an intracellular environment and, therefore, could be useful in the experimental exploration of biological signaling pathways—such as those crucial for oncogenic signal transduction. Additionally, this strategy may be suitable for therapeutic intervention to address a variety of diseases.

RNA-PROTACs

RNA-PROTACs are new chimeric structures that mediate the proteasomal degradation of RNA-binding proteins (RBP). They comprise an E3-recruiting peptide conjugated to a short-modified RNA, which is iso-sequential with the native binding element to which the RBP binds in cells. RNA-PROTACs offer a fast, rational, and general approach to targeting an entire class of protein (RBPs) that has proven difficult until today to target pharmacologically.

Oligonucleotide-based PROTAC

Transcription factor-targeting chimeras (TRAFTACs) consist of a chimeric oligonucleotide that binds to both the transcription factor of interest (TOI) and HaloTag-fused dCas9 protein, which can induce TOI degradation. Oligonucleotide-based PROTACs (O'PROTACs) not only can serve as a research tool but also can be harnessed as a therapeutic arsenal to target DNA binding proteins for effective treatment of diseases such as cancer.

CLIPTACs (In-cell click-formed proteolysis targeting chimeras)

Conventional PROTAC has a high molecular weight, which may limit its cellular permeability and solubility. CLIPTAC (in-cell click-formed proteolysis-targeting chimeras) was developed to overcome the limitation. The main potential advantage of CLIPTAC is a significant reduction of the molecular weight and polar surface area of the separate reaction partners compared to the pre-assembled PROTAC molecule.

Although the traditional PROTACs are expected to become an important method for certain target classes, some POIs do not possess the required small molecule binding sites. The emergence of alternative PROTAC technologies can overcome some limitations of the classical PROTAC model. Many emerging PROTAC-based technologies such as RNA-PROTACs and Photoswitchable PROTACs have also expanded the scope of PROTAC development, providing more feasible potential strategies for the clinical treatment of related diseases. Although they are in their infancy and may have some critical issues that need to be resolved, they pointed out the main direction of targeted protein degradation in the future.