Proteolysis Targeting Chimera (PROTAC): A Paradigm-Shifting Approach in Small Molecule Drug Discovery

Introduction

Significant progress has been made in small molecule drug discovery, particularly in targeted therapies for diseases such as cancer. Traditional approaches rely on inhibiting target protein activity to induce cancer cell death. However, tumor cells frequently develop resistance through overexpression or mutation of the target protein, restoring its function despite inhibition.

An emerging approach bypasses direct inhibition by using small molecules to degrade target proteins. This method utilizes the ubiquitin-proteasome system (UPS), which eliminates damaged or unneeded proteins. Specialized molecules, known as proteolysis targeting chimeras (PROTACs), exploit this system by tagging target proteins for degradation. These bifunctional molecules bind both the target protein and an E3 ligase, facilitating ubiquitination and subsequent proteasomal degradation. This strategy disrupts protein function at its source, potentially halting tumor proliferation.

Mechanism of PROTACs

PROTACs are heterobifunctional molecules composed of three parts: an E3 ligase-binding moiety, a target protein-binding moiety, and a linker connecting the two. This structure enables PROTACs to form a ternary complex involving the E3 ligase and the target protein, resulting in ubiquitin transfer and protein degradation. The ability to recycle and induce multiple degradation cycles distinguishes PROTACs from traditional inhibitors.

Applications and Developments

Since the introduction of the first small molecule PROTAC, various E3 ligases, including MDM2, cIAP1, cereblon, and von Hippel-Lindau (VHL), have been harnessed. PROTACs have proven effective against diverse protein targets, including transcription factors such as androgen receptor (AR), estrogen receptor (ER), and estrogen-related receptor alpha (ERRα). Degraders targeting RIPK2 and TBK1 have shown high specificity and anticancer activity in both wild-type and mutant cell lines.

A notable advancement involves BRD4-targeting PROTACs, such as dBET1 and ARV-825, which demonstrated enhanced downregulation of c-MYC and associated pathways. ARV-771 induces apoptosis in prostate cancer models, while MZ1 selectively degrades BRD4 over other BET family proteins. Structural studies of MZ1 revealed extensive protein-protein interactions within the ternary complex that drive specificity. Compound BETd-260/ZBC260, with picomolar potency, achieves complete tumor growth inhibition in preclinical models. Tyrosine kinases such as ALK, c-Abl, BTK, and PTK2 are also susceptible to PROTAC-induced degradation. BTK degraders like MT-802 and P131 are particularly promising for treating ibrutinib-resistant leukemia harboring the C481S mutation.

Advantages of PROTACs

PROTACs offer several key advantages over traditional small molecule drugs. First, their degradation-based mechanism allows for lower dosing and improved efficacy, as they do not require continuous occupancy of the active site. Second, selectivity is enhanced through the differential activity of E3 ligases and the unique surface interactions in the ternary complex. Third, PROTACs can potentially overcome resistance associated with mutations in target protein active sites, as they do not rely on inhibition of catalytic function.

Challenges and Considerations

Despite their potential, PROTACs face challenges. Their typically high molecular weight and polar surface area limit solubility, stability, and cell permeability, affecting pharmacokinetic properties. Innovative strategies such as in-cell self-assembly of PROTACs may help address these issues. Blood-brain barrier penetration remains limited, restricting applications in central nervous system diseases.

Additionally, although over 600 human E3 ligases exist, only a few have suitable ligands identified. Further research is needed to discover ligands with optimal affinity and safety profiles. The chemical linker between E3 and target ligands also critically affects degradation efficiency and pharmacokinetics. Guidelines for linker optimization are not yet well established. Finally, understanding the biological function of target proteins is essential to ensure effective and safe degradation.

Conclusion

PROTAC technology represents a transformative shift in drug discovery, offering the potential to target previously “undruggable” proteins. With improved selectivity, potency, and the ability to circumvent traditional resistance mechanisms, PROTACs hold promise for diverse therapeutic applications. Addressing pharmacokinetic limitations and expanding the repertoire AU-15330 of usable E3 ligases will be crucial for translating this approach into effective clinical treatments.