Expanding Undruggable Protein Regulation Pathways

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Undruggable proteins typically represent a class of proteins with large and complex structures or functions that are challenging to interfere with using conventional drug design strategies. Targeting such proteins is currently considered a potential opportunity for treating human diseases. Protein regulation can be broadly categorized into two major classes: protein degradation and protein stabilization. In recent years, significant progress in treating diseases through the direct degradation of proteins using Proteolysis-Targeting Chimeras (PROTACs) has underscored the importance of protein modulation drugs.

Targeted Protein Degradation Based on Proteasomes

The core mechanism of PROTAC action involves degrading target proteins through the ubiquitin-proteasome system (UPS). As a bifunctional molecule, PROTAC simultaneously targets the protein of interest and E3 ubiquitin ligase, forming a ternary complex. In this complex, the target protein is ubiquitinated, facilitating its recognition by the proteasome and subsequent degradation. Compared to traditional small molecule inhibitors, PROTAC utilizes an "event-driven" degradation process rather than an "occupancy-driven" binding mechanism, making PROTAC efficient in rapidly degrading intracellular target proteins, including drug-resistant mutants. PROTAC technology has the potential to degrade nearly 80% of human intracellular proteins and finds extensive applications in various fields, most notably in cancer therapy.

Fig. 1 PROTACs act through event-driven pharmacology. (Cecchini, 2021)

• Molecular Glue

Molecular glues have a dual-ligand structure targeting both E3 ubiquitin ligase and the target protein, enabling targeting protein-protein interactions (PPIs). Molecular glues feature smaller molecular weights, simpler chemical mechanisms, fewer spatial interferences, and superior drug formation. However, they cannot be designed through large-scale component screening as effectively as PROTACs. The molecular glue approach effectively overcomes limitations of traditional inhibitors, rendering previously undruggable targets druggable.

• p-PROTAC

Peptides, with their unique physicochemical properties, bridge the gap between small and large molecules, allowing small molecule drugs to access challenging drug-binding sites and helping alleviate resistance associated with small molecule drugs. Currently, most PROTACs use small molecules as targeting warheads, heavily relying on the binding pocket of the Protein of Interest (POI). The design of peptide-based PROTAC (p-PROTAC) aims to achieve specific and efficient degradation of POIs by employing peptides with high affinity for the POI binding site. This approach expands the range of non-druggable proteins. Simultaneously, it avoids limitations associated with shallow binding pockets, enhancing the substantial interaction surface between the POI and the peptide.

• CHAMP

Chaperone-mediated protein degradation (CHAMP) is another proteasome-based protein degradation technology utilizing molecular companions. CHAMP's structure is similar to PROTAC, with one end being the POI ligand and the other end being the molecular companion connected by a suitable linker. Similar to PROTAC, CHAMP facilitates the ubiquitination of the POI and its subsequent degradation by the proteasome.

Targeted Protein Degradation Based on Lysosome

Lysosomes and proteasome systems are the two main protein degradation pathways within cells, each with distinct functions. The proteasome system primarily degrades short-lived, soluble, and misfolded monomeric proteins, while the lysosome system mainly degrades long-lived proteins, protein aggregates, and damaged organelles. Both systems can achieve substrate degradation through ubiquitination and can collaboratively degrade the same intracellular substrates. Leveraging the lysosomal pathway for protein degradation allows the targeting of a broader range of substrates, including traditionally non-targetable proteins. The field currently comprises two main technologies: autophagy-lysosome pathway (e.g., AUTAC, ATTEC, and AUTOTAC) and endocytosis-lysosome pathway (e.g., LYTAC and MoDE-As).

Fig. 2 Several mechanisms of targeted proteins regulation strategies. (Xie, 2023)

• ATTEC

Autophagosome-tethering compound (ATTEC) is a compound that can simultaneously bind to LC3 and pathogenic biomolecules or organelles. ATTEC can tether the latter to the autophagosome through LC3, facilitating their autophagy-induced degradation. Due to the broad specificity of autophagic substrates, ATTEC is expected to expand the applicability of degraders to non-protein biomolecules or organelles.

• AUTAC

Autophagy-targeting chimera (AUTAC) is a degradation technology that utilizes autophagy by hijacking selective autophagy receptor SQSTM1/p62. Similar to PROTAC, AUTAC induces degradation through ubiquitination. However, PROTAC requires polyubiquitination at the K48 site of the POI for recognition by the 26S proteasome and subsequent degradation, while AUTAC requires triggering K63 polyubiquitination of the target to induce degradation. The nucleotide 8-nitro-cGMP (8-nitrocyclic guanosine monophosphate) serves as a crucial signaling molecule regulating autophagosome recruitment in cells.

• AUTOTAC

Autophagy-targeting chimera (AUTOTACs) is another degradation technology involving SQSTM1/p62. Unlike AUTACs, which require inducing target ubiquitination for degradation, AUTOTACs directly interact with the ZZ domain of SQSTM1/p62, without the need for polyubiquitination. Autophagy-targeting ligand (ATL) has been developed specifically for synthesizing AUTOTACs.

• LYTAC

Lysosome-targeting chimaeras (LYTACs) are bifunctional molecules with two binding domains: one end has a ligand that binds to the cell surface transmembrane receptor CI-M6PR (cation-independent mannose-6-phosphate receptor), and the other end has a POI ligand. These two binding domains are linked by a chemical connecting group. The trimeric CI-M6PR–LYTAC–POI complex formed on the cell membrane is engulfed, creating a transport vesicle. The vesicle transports the complex to the lysosome, where the POI is subsequently degraded.

• PROTAB

The PROTAB protein degradation technology platform employs a dual-specificity antibody approach, comprising a binding domain (anti-tag) targeting the transmembrane E3 ubiquitin ligase such as RNF43 or ZNRF3 and a binding domain (anti-POI) targeting the protein of interest. In a study, PROTAB effectively degraded insulin-like growth factor 1 receptor (IGF1R) in colorectal cancer patients. Researchers also demonstrated the platform's potential by degrading Her2 and PD-L1 targets.

• MoDE-As

Molecular degraders of extracellular proteins through the asialoglycoprotein receptor (MoDE-As) mediate the degradation of extracellular proteins. MoDE-A facilitates the formation of a trimeric complex between the POI and the asialoglycoprotein receptor (ASGPR) on liver cells. Subsequently, the POI undergoes lysosomal protease endocytosis and degradation.

Targeted Protein Stabilization (TPS)

Inspired by PROTAC, Targeted deubiquitinase-chimeras (DUBTAC) targeting TPS have been developed. Researchers combined EN523 (a molecule selectively binding to the OTUB1 enzyme) with lumacaftor, resulting in NJH-2-057. Compared to using lumacaftor alone, DUBTAC effectively inhibits ΔF508-CFTR degradation, enhances protein levels, and demonstrates better stability in cystic fibrosis patient-derived cells. Building on this, researchers developed a TF-DUBTAC, selectively stabilizing oncogenic proteins, including FOXO3A, p53, and IRF3. They chose a Foxo-specific DNA motif as a binding ligand and introduced an azide group at the 5' end of the DNA motif. Using the Click reaction, they connected EN523-BCN and N₃-FOXO-ODN to generate TF-DUBTAC.

Design of Degrader Conjugates

Various types of molecules (including antibodies, drugs, and small molecules) have been combined into a single chemical entity to create diverse conjugates with desired characteristics, such as effective delivery functionality and cytotoxic activity. This provides an attractive approach for the rational design of potential compounds targeting undruggable proteins. Various types of degrader conjugates, including PROTAC-antibody conjugates and LYTAC-antibody conjugates, have been designed to target undruggable proteins.

References

1. Cecchini, C., et al., From Conception to Development: Investigating PROTACs Features for Improved Cell Permeability and Successful Protein Degradation, Front. Chem., 2021, 9.
2. Xie, X., et al.,Recent advances in targeting the “undruggable” proteins: from drug discovery to clinical trials, Signal Transduction and Targeted Therapy, 2023, 8, 335.