PROTACs, a novel therapeutic approach, have garnered interest from numerous pharmaceutical companies, including Merck, Genentech, Pfizer, Novartis, Arvinas, C4 Therapeutics, Haisco Pharmaceutical, Kintor Pharmaceutical, and BeiGene.
Currently, approximately 20 PROTACs are in clinical development, with Arvinas’ ARV-471 advancing most rapidly and in Phase 3 clinical trials. Most PROTACs entering clinical trials are designed with CRBN ligands due to their smaller molecular weight and ease of optimization for drug-like properties. However, CRBN ligands are prone to off-target effects, leading to the degradation of neo-substrates. To enhance the selectivity of PROTACs based on CRBN ligands, structural modifications have been made to CRBN ligands, including the introduction of methoxy substitutions and alterations in linking positions and types.
Design of PROTACs
Proteolysis Targeting Chimeras (PROTACs), also known as bifunctional degraders, represent an emerging therapeutic approach. PROTACs consist of a ligand for the target protein, a small molecule ligand for E3 ubiquitin ligase, and a linker. PROTACs recruit E3 ubiquitin ligase to form a ternary complex with the target protein, inducing ubiquitination of the target protein and subsequent proteasomal degradation.
The design of PROTACs can be described as a linear system with four stages, designed in sequence based on specific objectives for each step. The first stage is the identification of a selective target binding moiety (TBM), the second stage involves optimizing TBM activity and selectivity, the third stage evaluates the E3 ligase binding moiety (EBM), and the fourth stage focuses on optimizing the linker. Based on the results of each stage, an iterative process can be employed to refine early design elements.
The discovery of PROTACs holds the promise of overcoming some limitations of traditional small molecule inhibitors. PROTAC-induced degradation results in the formation of a ternary complex with ubiquitination capability, independent of directly blocking an active site. Consequently, PROTACs can be used to degrade target proteins that have poor efficacy with small molecule binding ligands, expanding the druggable target space. PROTACs also exhibit catalytic activity, eliminating the need for prolonged occupancy, as required by small molecules for therapeutic effects. Moreover, PROTACs degrade entire proteins, disrupting both enzymatic and non-enzymatic functions (e.g., scaffolding or transcriptional functions), as opposed to merely inhibiting protein activity, potentially overcoming resistance seen with traditional small molecules.
Off-Target Effects of CRBN
E3 ubiquitin ligase CRBN has been widely used in degrader design, with common CRBN ligands including immunomodulatory drugs such as thalidomide, lenalidomide, and pomalidomide.
Compared to VHL, CRBN ligands have smaller molecular weights, making them more amenable to optimization for drug-like properties. Consequently, most PROTACs entering clinical trials are based on CRBN ligand design. However, they often exhibit selectivity issues.
Immunomodulatory drugs bind to a hydrophobic pocket at the C-terminus of CRBN protein. CRBN ligands can act as molecular glue on CRBN, inducing conformational changes on the surface and recruiting, ubiquitinating, and subsequently degrading some neo-substrates, such as Ikaros (IKZF1), Helios (IKZF2), Aiolos (IKZF3), SALL4, and GSPT1. Therefore, the use of CRBN ligands in degraders commonly results in off-target effects, lacking selectivity for the target protein.
While in some cases, degrading target proteins and neo-substrates may be beneficial, degrading neo-substrates can lead to undesirable toxicities. For example, Ikaros and Aiolos are essential for lymphocyte development, and the degradation of SALL4 is associated with teratogenicity. ZFP91 has been shown to be crucial for T cell function. The degradation of neo-substrates is attributed to the binding of the phthalimide group of CRBN ligands within the thalidomide binding site of CRBN. Subsequently, an indole or phthalimide forms a new interaction with a critical recognition sequence, termed a structural degron, which contains an essential glycine residue in a β-turn loop.
Many neo-substrates are zinc-finger transcription factors containing G-loop degrons, which are challenging to target with traditional small molecules due to the lack of suitable binding pockets. Some neo-substrates, not being zinc-finger transcription factors, still contain G-loop degrons. GSPT1 is one such example. Despite the ongoing clinical trials of GSPT1 degraders for the treatment of acute myeloid leukemia, knocking out GSPT1 results in widespread cytotoxicity due to its essential cellular functions. Off-target degradation of GSPT1 may reduce the therapeutic index, especially in non-oncology indications.
It is because CRBN ligands can lead to off-target effects that an increasing number of research groups and companies are focusing on enhancing CRBN selectivity. Co-crystal structures suggest that the glutarimide ring of pomalidomide is deeply buried within the CRBN protein, with only the phthalimide ring being amenable to modification.
Reducing Off-Target Effect through CRBN Ligand Modification
- Methoxy-Substituted CRBN Ligands
A research group reported a class of thalidomide derivatives that do not degrade neo-substrates but can still be used in degrader design to target specific proteins.
They first presented CC-885 as an example. CC-885 can degrade GSPT1, IKZF1, and IKZF3. By introducing a methoxy group at the 7th position of the benzene ring, they obtained compound 2, which retained similar CRBN binding capability as CC-885 but blocked the degradation of neo-substrates. Compound 2, as shown in Figure 3, does not degrade GSPT1, IKZF1, or IKZF3 proteins.
Subsequently, they employed these CRBN ligands for the design of BRD9 degraders. They designed degraders using pomalidomide as the CRBN ligand (compound 15) and methoxy-substituted pomalidomide as the CRBN ligand (compound 16). As shown in Figure 4, both compounds exhibited good CRBN binding capability and could effectively degrade BRD9. However, compound 16 demonstrated better selectivity, not degrading neo-substrates. Proteomics experiments confirmed that only compound 16 selectively degraded BRD9.
- Changes in Substituent Type and Position
Researchers in the United States analyzed the degradation of GSPT1 and IKZF1 by 14 closely related thalidomide derivatives. They extensively explored the impact of substituents on activity and found that 4-hydroxy substitutions in both thalidomide and indolinone moieties did not induce degradation. However, 4-hydroxy substitution in isoindolinone resulted in IKZF1 degradation.
5-hydroxy substitutions in isoindolinone and 5-hydroxy substitutions in phthalimide induced GSPT1 degradation without affecting IKZF1. Among them, 5-hydroxy substitution in phthalimide demonstrated stronger GSPT1 degradation than 5-hydroxy substitution in isoindolinone, highlighting the critical role of 5-position substitutions for GSPT1 degradation.
5-amine substitutions in phthalimide and indolinone did not induce GSPT1 degradation and minimally affected IKZF1 degradation. No degradation occurred with 6- and 7-position substitutions. These findings can also guide the design of PROTACs to avoid off-target effects.
- Fewer Hydrogen Bond Donors and 5-Position Modification
Researchers published a similar report on bioRxiv. They discovered that specific substitutions at position C5 significantly reduced the tendency for neo-substrate degradation.
Substitutions at 4th and 5th positions, which contain hydrogen bond donors, could induce degradation of zinc-finger domains. They subsequently modified positions 4 and 5, synthesizing a series of compounds. The same chemical structure modification revealed that 5-position substitutions were more effective at reducing zinc-finger degradation than 4-position modifications, possibly due to better steric hindrance against zinc-finger domain formation.
Their structural modifications also underscored the importance of having an aromatic amine linker in pomalidomide-based PROTACs, where NH- serves as a hydrogen bond donor, inducing zinc-finger degradation. PROTACs lacking hydrogen bond donors exhibited minimal off-target activity. These findings align with previous reports. By adding a fluorine group at the C6 position, they further reduced zinc-finger degradation.
Finally, the authors provided two primary rules for designing pomalidomide-based PROTACs to minimize off-target effects. First, select the C5 position for the linker attachment. Second, hydrogen bond donors should not be connected to the phthalimide ring.
Similarly, a Japanese research group published similar work. They demonstrated the critical role of modifying the 6th position of lenalidomide in controlling neo-substrate selectivity, leading to selective degradation of IKZF1, IKZF3, and CK1α involved in hematological malignancies, and displayed stronger antiproliferative effects on multiple myeloma (MM) and myelodysplastic syndrome (MDS)-derived cell lines than lenalidomide. BET degraders based on 6th position-modified lenalidomide exhibited the same neo-substrate selectivity.
In conclusion, thalidomide and thalidomide derivatives consist of two chemical rings: a phthalimide and a glutarimide ring. The former binds to the C-terminal region of the CRBN protein, while the latter induces neo-substrate degradation. Chemical modifications to the phthalimide ring are crucial for the selectivity of neo-substrate degradation.
References:
- Laramy, M. N., et al., Bartlett, Delivering on the promise of protein degraders, Nature Reviews Drug Discovery, 2023, 22, 410-427.
- Bouguenina, H., et al., A degron blocking strategy towards improved CRL4CRBN recruiting PROTAC selectivity, ChemBioChem, 2023, e202300351.
- Nowak, R. P., et al., Structural rationalization of GSPT1 and IKZF1 degradation by thalidomide molecular glue derivatives, RSC Medicinal Chemistry, 2023, 14 (3), 501-506.
- Nguyen, T. M., et al., Proteolysis Targeting Chimeras With Reduced Off-targets, bioRxiv, 2023, 2021.11.18.468552.