📄 chemistry

Reversible covalency reprograms CRBN substrate recruitment and PROTAC-mediated degradation

This study demonstrates that boronic acid-based IMiD derivatives engage the CRBN E3 ligase via reversible covalent coordination with His353, a mechanism that reshapes substrate recruitment and enables the development of highly efficacious PROTACs with sustained degradation capabilities.

Original authors: Ivan Dikic, Henry Bailey, Thomas Geiger, Serhii Zhersh, Nataliya Tolmachova, Vinay Singh, Ina Dressel, Rubina Kazi, Jonathan Eisert, Jan Gerhartz, Dominika Pieńkowska, Lina Read, Se-Hun Kim, Maksym Bu

Published 2026-06-24

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CC BY 4.0

Original authors: Ivan Dikic, Henry Bailey, Thomas Geiger, Serhii Zhersh, Nataliya Tolmachova, Vinay Singh, Ina Dressel, Rubina Kazi, Jonathan Eisert, Jan Gerhartz, Dominika Pieńkowska, Lina Read, Se-Hun Kim, Maksym Bugera, Oleg Michurin, Tim Schober, Yuliia Mamchych, Nataliia Shliakhtova, Justyna Adamczyk, Petro Borysko, Urbi Mukhopadhyay, Jessica Fechner, Ulrike Bauer, Birgitta Leuthner, Thorsten Mosler, Fiona Sorrell, Frederik Lermyte, Radosław Nowak, Ivan Kondratov

Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). ✨ This is an AI-generated explanation of the paper below. It is not written or endorsed by the authors. For technical accuracy, refer to the original paper. Read full disclaimer

The Big Idea: A "Reversible Velcro" for Protein Cleanup

Imagine your body is a busy city, and inside every cell, there is a specialized trash collection service. This service is run by a machine called the E3 ligase (specifically, a part of it called CRBN). Its job is to tag unwanted or broken proteins with a "trash tag" (ubiquitin) so the cell's recycling plant can destroy them.

Scientists have developed a way to trick this trash machine into destroying specific "bad" proteins that cause diseases. They do this using a tool called a PROTAC. Think of a PROTAC as a double-sided glue stick:

One side sticks to the trash machine (CRBN).
The other side sticks to the bad protein you want to destroy (like BRD4).
When both sides stick, the trash machine grabs the bad protein and throws it away.

The Problem: The "Hook" and the "Permanent Glue"

Usually, these glue sticks are very strong, but they have two problems:

The Hook Effect: If you use too much glue, the trash machine gets overwhelmed. It ends up sticking to the glue stick itself instead of the bad protein, and the trash collection stops working.
Permanent Glue: Some scientists tried making the glue stick "irreversible" (like super-glue) so it never lets go. While this makes it stick better, it can be messy. It might get stuck in the wrong place or stop the machine from working efficiently because it's too rigid.

The New Solution: "Reversible Velcro" (Boron)

This paper introduces a new type of glue stick made with a special ingredient called Boron.

Instead of using super-glue (permanent) or weak tape (non-covalent), the scientists created a "Reversible Velcro."

How it works: The Boron acts like a smart magnet. It snaps onto a specific spot on the trash machine (a part called His353) very tightly, but it can also unclip if needed.
The Shape Change: When this Boron "Velcro" snaps onto the trash machine, it changes the machine's shape slightly. It's like putting a specific hat on the trash collector that locks its hands in a new position.

What Happened When They Tried It?

1. It Sticks Better, But Doesn't Get Stuck Forever
The scientists tested this new Boron glue on the trash machine. It stuck much tighter than the old non-covalent glue, but unlike the super-glue, it wasn't permanent. This gave them the best of both worlds: strong binding with the flexibility to let go when necessary.

2. It Changes What the Trash Machine Eats
Normally, the trash machine (CRBN) is known to eat a specific list of "bad proteins" (like IKZF1 and SALL4) when you give it a standard glue stick.

The Discovery: When the scientists used the new Boron "Velcro," the trash machine stopped eating that specific list of proteins.
Why? Because the Boron changed the shape of the machine's "mouth" (the binding pocket). It was like putting a different hat on the collector that made it unable to grab those specific items, even though the collector itself was still working.

3. It Makes a Better Trash Collector for Bad Proteins
The scientists then built a full PROTAC (the double-sided glue stick) using this new Boron Velcro to target a protein called BRD4.

The Result: This new tool was incredibly effective. It destroyed the BRD4 protein much more thoroughly than the old tools.
The "Hook" Fix: The old tools would stop working if you used too much of them (the "Hook Effect"). The new Boron tool kept working efficiently even at high doses. It was like having a trash collector that didn't get confused or overwhelmed, even when the street was full of trash.

4. A New Shape for the Team
When they looked at the molecular structure (like taking a 3D photo of the team), they saw something amazing. The Boron didn't just hold the trash machine and the bad protein together; it acted as a bridge. It touched the trash machine and the bad protein at the same time, creating a new, stable triangle shape that made the whole process much more efficient.

The Bottom Line

The researchers discovered a way to use Boron to create a "Reversible Velcro" that attaches to the cell's trash machine.

It makes the attachment stronger than normal glue.
It prevents the trash machine from grabbing the wrong things (changing its "menu").
It allows the trash machine to work more efficiently and for longer periods without getting confused or overwhelmed.

This proves that using Boron to create reversible, strong bonds is a powerful new strategy for designing better medicines that clean up specific bad proteins in the body.

Technical Summary: Reversible Covalency Reprograms CRBN Substrate Recruitment and PROTAC-Mediated Degradation

Problem Statement
Targeted protein degradation (TPD) via PROTACs and molecular glues has validated the degradation of previously "undruggable" proteins. While covalent ligands targeting E3 ubiquitin ligases (such as CRBN) can enhance ternary complex stability and degradation efficiency, irreversible covalent modification presents challenges regarding selectivity, toxicity, and the loss of catalytic turnover. Conversely, non-covalent ligands may lack sufficient residence time. There is a need for strategies that combine the high potency of covalent binding with the reversibility required for catalytic pharmacology, specifically to modulate the substrate recruitment landscape of the E3 ligase Cereblon (CRBN).

Methodology
The authors employed a multidisciplinary approach combining structure-guided chemical design, structural biology, biochemical assays, cellular degradation profiling, and global proteomics:

Chemical Design: A library of boronic acid-based IMiD derivatives (Bo-IMiDs) and corresponding PROTACs (Bo-TACs) was synthesized. These compounds feature pinacolboronate (BPin) or boronic acid (Bo) groups positioned to interact with the CRBN sensor residue His353.
Structural Biology: X-ray crystallography was used to solve binary (CRBN-ligand) and ternary (CRBN-ligand-PROTAC-substrate) complex structures. Native ion mobility mass spectrometry (IM-MS) and collision-induced dissociation (CID) were utilized to assess complex stability.
Biochemical & Cellular Assays: Fluorescence polarization (FP) and NanoBRET assays measured binding affinity and cellular target engagement. Washout experiments distinguished between reversible and irreversible covalent mechanisms. Degradation efficacy was quantified using HiBiT reporter assays for BRD4, IKZF1, and SALL4.
Proteomics: Global proteomics (HiBiT and mass spectrometry) and chemoproteomics (ProxiCapture) were performed in MOLT-4 cells to map the CRBN interactome and degradome, comparing Bo-IMiDs/Bo-TACs against canonical IMiDs (e.g., pomalidomide) and non-covalent controls.
Mathematical Modeling: Ternary complex formation and degradation kinetics were modeled to analyze the "hook effect" and cooperativity.

Key Contributions and Results

Discovery of Reversible Covalent CRBN Engagement:
The study identified that boronic acid-modified IMiDs (Bo-IMiDs) engage CRBN through a reversible covalent interaction with the imidazole side chain of His353. Crystal structures (1.7–2.2 Å resolution) revealed the formation of a tetrahedral boronate adduct between the boron center and the $\epsilon$ -nitrogen of His353. This interaction increases binding potency (6–18-fold improvement in IC50) compared to non-covalent or bromine-modified controls, while maintaining reversibility as confirmed by CID mass spectrometry (intermediate stability between non-covalent and irreversible covalent binders).
Reprogramming of Molecular Glue Activity:
Canonical IMiDs (e.g., thalidomide, lenalidomide) act as molecular glues by recruiting neo-substrates (IKZF1, GSPT1, SALL4) via a G-loop degron. The authors demonstrated that Bo-IMiDs completely abrogate the degradation of these canonical G-loop substrates. Structural overlay suggests that the covalent locking of His353 in an alternative rotamer sterically clashes with the G-loop binding site, effectively reshaping the CRBN interactome to exclude canonical molecular glue substrates.
Enhanced PROTAC Efficacy and Expanded Therapeutic Window:
Incorporating Bo-IMiDs into BRD4-targeting PROTACs (Bo-TACs) resulted in deep and efficacious degradation.
- Selectivity: Bo-TACs retained high-affinity BRD4 recruitment but suppressed the recruitment of off-target canonical molecular glue substrates (IKZF1, IKZF2, etc.), improving degrader selectivity.
- Hook Effect Mitigation: Despite exhibiting negative cooperativity in ternary complex formation, Bo-TACs displayed a delayed onset of the "hook effect" (degradation inhibition at high concentrations). Mathematical modeling and degradation curves showed that Bo-TACs maintained >99% degradation across a broader concentration range compared to unmodified or irreversible covalent PROTACs.
- Ternary Complex Stability: TR-FRET assays indicated that Bo-TACs form more stable ternary complexes with higher maximal FRET signals, suggesting improved conformational stability.
Structural Basis for Enhanced Degradation:
The crystal structure of the ternary complex (CRBN:Bo-TAC:BRD4) revealed that the boronic acid moiety acts as a "chameleonic" interaction center. Beyond covalently binding His353, the boronic acid hydroxyl groups form hydrogen bonds with the substrate BRD4 (specifically Gly143 and Asp145). This creates a novel interaction network involving additional protein-protein contacts (e.g., BRD4 Trp81 with CRBN His103) that were not observed in previous non-covalent PROTAC structures, likely contributing to the enhanced degradation efficiency.
Extension to Minimal Scaffolds:
The strategy was successfully applied to minimal CRBN ligand scaffolds (phenyl-glutarimide and phenyl-dihydrouracil). Boron substitution at specific positions (4-position of the phenyl ring) significantly improved affinity (shifting from $\mu$ M to nM range) while preserving favorable physicochemical properties (solubility, metabolic stability, and low molecular weight), demonstrating the generalizability of this approach for ligand optimization.

Significance
The paper establishes reversible covalent targeting of CRBN via His353 as a viable strategy to modulate E3 ligase function. By combining the potency of covalent binding with the controllability of reversible interactions, this approach:

Decouples high-affinity E3 engagement from canonical molecular glue substrate recruitment, allowing for the design of highly selective PROTACs.
Expands the chemical space for targeted protein degradation by introducing boron chemistry as a versatile pharmacophore that can simultaneously engage the E3 ligase and the substrate.
Improves the therapeutic window of PROTACs by mitigating the hook effect, enabling deep degradation across a wider concentration range.
Provides a blueprint for developing minimal, drug-like ligands with enhanced affinity through reversible covalent chemistry.

The authors conclude that this modality offers a new facet of boronate utility in drug discovery, enabling dynamic target engagement and the formation of novel interaction networks that enhance degradation productivity.