Novel Intramolecular Bivalent Glue Targets Degradation of BRD4

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Targeted protein degradation is an ideal pharmacological model for overcoming the limitations of traditional drug discovery techniques. Biodegradation methods based on ubiquitin-proteasome system are currently the most mature system, which can be mainly divided into two categories, one is proteolysis targeting chimeras (PROTACs), and the other is molecular glues. Both of them induce the proximity of E3 ubiquitin ligase to the target protein by drug molecules to catalyze the polyubiquitination modification of the target protein, which is then recognized and degraded by the proteasome.

In February 2024, Alessio Ciulli's research group from the University of Dundee published a research article on "Targeted protein degradation via intramolecular bivalent glues" in Nature. Through orthogonal genetic screening, biophysical characterization and structural reconstruction, we studied the bifunctional degraders of bromodomain-containing protein 4 (BRD4), and found a new strategy based on the ubiquitin-proteasome system - intramolecular bivalent glue (IBGs). The researchers found that IBGs did not bind the target protein and E3 ubiquitin ligase like traditional PROTAC, but simultaneously bound and linked two adjacent domains of the target protein, causing the conformation of the target protein to change, and then adhere to the E3 ubiquitin ligase DCAF11 or DCAF16.

Schematic model of the different modes of molecular recognition with traditional monovalent lues and bivalent PROTACs versus intramolecularly bivalent glues. (Hsia, O., 2024)

Research of IBG1

According to the patent published in 2021, researchers synthesized a PROTAC-like degrader, IBG1, which is linked by BET bromine domain inhibitor JQ1 and aryl sulfonamide molecular gel E7820, specifically degrading BRD4 and BRD2 in a variety of cancer cell lines. The results showed that the proteasome inhibitor MG132 and the ubiquitination inhibitor MLN4924 could block the degradation of BRD4 by IBG1, demonstrating the ubiquititation-proteasome degradation pathway. The molecular glue E7820 has been shown to induce ubiquitination and degradation of the RNA-binding protein RBM39 by recruiting the E3 ligase DCAF15. However, it was found that the degradation of BRD4 by IBG1 was independent of DCAF15.

IBG1 degrades BRD2 and BRD4 independently of DCAF15. a, Structure of IBG1. b, BET protein degradation activity of IBG1. (Hsia, O., 2024)

To identify the E3 ligase required for IBG1 degradation activity, the researchers performed a BRD4 degradation CRISPR screen. Similar to the reported DCAF16 molecular gel GNE-0011, the degradation of BRD4 by IBG1 is associated with the CRL4-DCAF16 complex, and the knockdown of both DCAF16 and DDB1 inhibits BRD4 degradation.

The potential affinity of IBG1 to DCAF16 was further investigated. The results show that IBG1 has no affinity for DCAF16, but part of E7820 has some affinity for DCAF15. Excessive E7820 and IBG1 analogues that retain part of E7820 do not prevent ubiquitination and degradation of BRD4, and IBG1 analogues with part of E7820 that have a simplified structure do not show BRD4 degradation activity. These results suggest that the E7820 portion of IBG1 is not involved in the recruitment of DCAF16, but is essential for its degradation activity.

The researchers observed the formation of ternary complexes of IBG1, DCAF16 and BRD4^Tandem using isothermal titration calorimetry and time-resolved fluorescence resonance energy transfer techniques. DCAF16 itself has an intrinsic affinity for BRD4^Tandem, and the addition of IBG1 enhances the interaction between the two. However, DCAF16 has no affinity for a single BRD4^BD1 or BRD4^BD2, and the addition of IBG1 does not cause an interaction. These results suggest that degradation may require the involvement of two bromine domains.

Further study showed that the deletion of other domains except BD1 and BD2 did not affect the degradation activity. In contrast to PROTAC dBET6, IBG1 does not degrade individual BRD4^BD1 and BRD4^BD2. The replacement of BRD4^BD1 with BRD2^BD1 and BRD3^BD2 does not affect degradation activity, nor does the replacement of BRD4^BD2 with BRD2BD2, but the replacement of BRD4^BD2 with BRD3^BD2 blocks degradation. These results suggest that the degradation of BRD4 by IBG1 requires the simultaneous participation of both bromine domains, and BD2 determines the selectivity of the degradation.

The structure of BRD4^Tandem, IBG1 and DCAF16 ternary complex was analyzed by cryo-electron microscopy. The JQ1 part of IBG1 binds to BRD4^BD2, the E7820 part binds to BRD4^BD1, and DCAF16 encloses hydrophobic dimethylthiophene and phenyl in the JQ1 part. DCAF16 binds to a hydrophobic pocket of BD1 via W54, and the conformational change caused by IBG1 binds to both bromine domains at the same time allows for an interaction between BD1 and BD2. Similar to the above results, BRD3-BD2^G386 and BRD4-BD2^G386 determine the selectivity of degradation. The results showed that IBG1 induced dimerization of target protein to degrade BRD4, which is a novel protein targeted degradation strategy.

Molecular glue at BOC Sciences

Design optimization for other IBGs

Based on the mechanism obtained by crystal structure analysis, the subsequent researchers used this idea to optimize the structure of IBG1, and replaced the E7820 part with the JQ1 part with higher affinity to design IBG2 and IBG3. The results showed that IBG3 selectively degraded BRD4 and BRD2 more efficiently and through the same mechanism as IBG1.

An intramolecular adhesive based on DCAF11

As an intramolecular binder based on DCAF11, the researchers speculate that bivalent domain binding via intramolecular binders may be widely used to modulate protein-protein interactions, thereby reconfiguring protein function. A patent from 2022 reports a structurally-similar selective BRD4 degrader, IBG4, that connects pyrazolopyrimidine groups to JQ1 via short, rigid connectors, similar to IBG1, showing effective degradation of BRD4^Tandem. The isolated bromine domain and acetyllysine pocket mutants N140F and N433F are also avoided. The degree of tandem compactness induced by IBG4 was similar to that of IBG1, and the NanoBRET conformational biosensor showed that both compounds induced intramolecular interactions in the bromine domain, indicating that IBG4, similar to IBG1, induced cis-dimerization of the bromine domain. The pyrazopyrimidine portion of IBG4 has a similar affinity to the BRD4 bromine domain as the E7820 portion of IBG1. Although structurally different, IBG4 mimics the role of IBG1 in cellular mechanisms, suggesting that the two compounds share an intramolecular binder-like mechanism. Unlike IBG1, IBG4 has a high specificity for BRD4 and fails to degrade BRD2 effectively, suggesting that the potential BRD4-IBG4-E3 ligase complex has different structural requirements. In fact, although neddylation inhibitor MLN4924 prevented degradation, DCAF16 knockout had no effect on IBG4-mediated BRD4 degradation. The results show that IBG4 generalizes all the cellular and biophysical properties of intramolecular glue degraders, but extends the mechanism of action to another structurally unrelated E3 ligase.

The future of intramolecular bivalent glue

To date, most molecular gel degraders work by binary binding to E3 ubiquitin ligase, which then recruits substrate proteins for ubiquitination modification. Therefore, it is impossible to carry out reasonable design, which greatly limits the number of molecular glue class degraders. In this study, by binding IBGs to two bromine binding domains on BRD4, the intramolecular dimerization occurs to form a new protein conformation, which then forms a stable ternary complex with E3 ubiquitin ligase and induces the degradation of the target protein. About 60-80% of human proteins have at least two distinct domains, so it is possible to target degradation through intramolecular dimerization, and more specific targeted protein degradation drugs are expected to be designed based on such methods.

References:

1. Hsia, O., et al. Targeted protein degradation via intramolecular bivalent glues. Nature. 2024, 627(8002): 204-211.
2. Du, X., et al. Structural basis and kinetic pathway of RBM39 recruitment to DCAF15 by a sulfonamide molecular glue E7820. Structure. 2019, 27(11): 1625-1633.