• Expanding the Toolbox of PROTAC Degraders with Covalent Ligands for Genetically Validated Targets

    Illustration of the ubiquitin-proteasome system tagging and degrading a protein, a process similar to PROTAC drug action

    Posted on May 22, 2023

    What are PROTAC degraders or protein degrader drugs?

    Proteolysis Targeting Chimeric (PROTAC) drugs are a type of novel heterobifunctional molecules that utilize the ubiquitin-proteasome system to selectively degrade specific proteins in cells. Typical PROTAC drugs are composed of a ligand for the protein-of-interest (POI) or target protein and a ligand for E3 ubiquitin ligase, where both ligands are linked by a chemical linker (Fig. 1). PROTAC therapy involves linking a protein of interest to an E3 ligase enzyme, which facilitates the ubiquitination of the protein. Once the protein is tagged with ubiquitin, it is targeted for degradation by the proteasome, a cellular machinery responsible for protein degradation. This targeted protein degradation approach can be used to directly target harmful proteins, such as those involved in tau-protein diseases or viruses, or to target protein complexes that inhibit specific downstream activities. Several PROTAC small molecules are currently in clinical trials with promising results, indicating the potential of this technology for therapeutic applications.

    Fig. 1 PROTAC Strategy

    We offer custom-tailored cell-based assays, lot release potency assays and stability studies for PROTAC drugs in compliance with cGMP standards.
    Speak to a scientist to learn more.

    Reversible non-covalent PROTAC vs. Irreversible covalent PROTAC

    Over the past two decades, chimeric degraders have gone from being chemical biology tools to clinical assets. However, most chimeric degraders still focus on well-validated ligandable targets, leaving a gap in the field for genetically validated proteins that lack small molecule binding sites. One approach to address this challenge is the development of covalent ligands, which can target a cryptic pocket and partially compensate for limited non-covalent binding affinity. Incorporating these ligands as components of protein degraders has become increasingly popular. Irreversible covalent degraders may provide an attractive approach against some targets. In a recent study, an irreversible-covalent degrader (BCCov) of BTK as the target and cIAP1 as the E3 ligase was investigated. Bruton’s Tyrosine Kinase (BTK) is a therapeutically relevant protein that presents a reactive cysteine (C481) in the ATP binding pocket of the kinase domain. BTK is a validated target for B-cell malignancies, and several molecules targeting BTK have progressed to Phase 1 clinical trials. However, there is uncertainty around the consequences and context of covalent modification of BTK. Finally, there are few structural insights into the formation of a productive target-degrader-E3-ligase ternary complex, with no examples yet representing a covalent assembly. Despite its wide utility in the protein degrader field, the cellular inhibitor of apoptosis (cIAP1) remains limited in terms of deep mechanistic characterization.

    Orthogonal studies of covalently modified BTK

    This study expands understanding of target recruitment within the framework of covalent degraders targeting BTK and co-opting cIAP1. The results show that covalently modified BTK is a bonafide target for proteasomal degradation and represents yet another unique ternary complex pose for this protein combination. This research can pave the way for further development of covalent degraders for therapeutic purposes. Although covalent modification of BTK does not seem to be necessary for its degradation by BCCov, BCCov can still covalently modify BTK in cells. To test whether covalent modification precedes BTK degradation in the cellular context, parallel time-course studies were performed. In one assay, BTKFL is con-transfected with cIAP1FL, treated it with 5 µM BCCov, and measured degradation over time by western blot. In the second assay, we transfected BTKFL in the absence of exogenous cIAP1, treated it with an equivalent amount of BCCov, and analyzed it by mass spectrometry at matched intervals. The degradation was observed starting as early as 30 min and reaching a plateau by the 7-hour timepoint. Intriguingly, covalent modification occurs on a similar, though slightly slower time scale, suggesting that covalently modified BTK may form during the degradation time course.

    After conducting cellular experiments, interactions between BTK, BCCov, and cIAP1 were examined using an in vitro reconstituted system. The ternary complex formed between BTKBCCov, cIAP1BUCR1, and [cIAP1BUCR1-BCNC] had an approximate 2:2 or 1:2 stoichiometry and showed no fundamental defect in ternary complex formation and ubiquitin transfer. A relatively tight ternary complex with a Kd of 72.5 nM, which is similar to the affinity of a [cIAP1BUCR1-BCNC] binary complex binding BTKKD, suggesting that the cIAP1 affinity is not substantially impacted by the BTK~BCCov covalent bond. The affinity of the ternary complex remains relatively high but weaker than the binary affinity measured against cIAP1 alone. The cooperativity factor for BCCov was calculated to be ≤0.2, indicating a relatively tight, yet negatively cooperative ternary complex.

    Structural studies of the BTK covalent ternary complex

    Structural studies were conducted in order to gain deeper insights into the topology and interactions of the BTK covalent ternary complex. A 3.0 Å (2.3 Å anisotropic) structure of the ternary complex revealed a distinct ternary complex pose for this target/E3 ligase pair and showed that the covalent attachment of BCCov to Cysteine 481 of BTK is unambiguously resolved. Although the electron density map was modest, conformational changes in BTK that differed from the previously reported ibrutinib-bound structure were observed, including a striking outward flip of F559. The resulting contacts in this non-native assembly highlighted the flexibility and induced contacts in the ternary complex. Despite the apparent favorable interaction between F559 and BCCov, this contact was found to be dispensable for BCCov-mediated BTK degradation. Finally, a series of intimate lipophilic contacts between BTKKD and cIAP1Bir3 were observed, including several proximal to the F559/BCCov interaction with alternating residues zippering at the interface.

    In order to confirm the results obtained from crystallography and investigate the structure of BTKKD-BCCov-cIAP1Bir3 in solution, 2-dimensional (2D) NMR experiments were conducted. First generated a cIAP1Bir3-BCCov binary complex by pre-incubating uniformly [13C,15N]-labelled cIAP1Bir3 with BCCov. Then, BTKKD is added to the binary complex to create a ternary complex. The addition of BTKKD resulted in perturbations in residues located in the C-terminal helices and preceding loops, which is consistent with the interface observed in the crystal structure. Furthermore, severe line-broadening for a specific set of residues near the cIAP1 binding site were obsereved, indicating slow conformational exchange on the μs-to-ms time scale and potentially reflecting multiple binding modes of BCCov to cIAP1Bir3.


    The researchers established an engineered cellular system to investigate the distinct steps of the process, confirming the ability of BCCov to covalently modify BTK in cells. They also confirmed that covalently modified BTK is compatible with proteasomal degradation. In addition, they established a suite of in vitro assays to characterize the biophysics and structure of BTK~BCCov recruitment to the E3 ligase cIAP1. Finally, the researchers observed an interesting phenomenon with partial hook effect for the pairing that permits covalent BTK recruitment, suggesting the potential for greater potency with covalent irreversible degraders. However, the possible in vivo consequences of this effect remain unclear due to the dynamic nature of dosing, distribution, and metabolism. Overall, this study provides insights into the potential of covalent ligands as a new approach to targeted protein degradation.

    “Parts of the figure were drawn by using pictures from Servier Medical Art. Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/).”