Introduction
Targeted protein degradation (TPD) is accomplished by the induced proximity of an E3 ubiquitin ligase and a target protein to promote target ubiquitination and subsequent proteasomal degradation. TPD is an emerging therapeutic tool with high potential to tackle disease-causing proteins. In the past, TPD has been achieved either by using PROteolysis-TArgeting Chimeras (PROTACs)—bifunctional compounds composed of two separate moieties that individually bind the target and E3 ligase, or via molecular glues that monovalently bind either the ligase or the target. Herein, we highlight the mechanism of action of novel bifunctional degraders of BRD4, termed Intramolecular Bivalent Glues (IBGs), introducing a new modality in targeted protein degradation. Instead of connecting target and ligase in trans as PROTACs do, IBGs simultaneously engage and connect two adjacent domains of the target protein in cis, thereby enhancing surface complementarity with E3 ligases. This conformational change ‘glues’ BRD4 to the E3 ligase DCAF16, utilising the intrinsic target–ligase affinities, leading to the degradation of BRD4 that does not occur in the absence of compound. Structural insights into the ternary BRD4–IBG1–DCAF16 complex guided the rational design of further improved degraders of low picomolar potency.
Assay principle
Ternary complex formation involving BRD4 and DCAF16 is evaluated with a Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) complex-formation assay. This is achieved by using a europium-labelled antibody against BRD4 and Cy5-labelled DCAF16. If an IBG molecule induces the formation of a ternary complex between the labelled BRD4 and DCAF16 molecules, the fluorophores are in sufficient proximity to form a FRET pair. Thus, exciting the europium donor fluorophore will result in energy transfer and Cy5 acceptor fluorescence can be measured with a microplate reader.
Materials & methods
- White 384-well plate
- Sulfo-Cy5 labelled DCAF16
- His-tagged BRD4 constructs
- anti-His-europium donor (PerkinElmer)
- PHERAstar FS (BMG LABTECH)
Experimental Procedure
TR-FRET-based molecular glue binding assays
Stock solutions of sulfo-Cy5-labelled DCAF16, His-BRD4 and anti-His-europium donor were prepared in TR-FRET assay buffer (50 mM HEPES pH 7.5, 100 mM NaCl, 1 mM TCEP, 0.05% Tween-20). Two types of TR-FRET assays were performed: titration of IBGs into a mix of BRD4 and Cy5-DCAF16 (complex-formation assay) and titration of sulfo-Cy5-labelled DCAF16 into BRD4 or BRD4 + IBG (complex-stabilisation assay). For the former, compounds were titrated 1:4 into 100 nM BRD4 and 100 nM Cy5-DCAF16 to a white 384-well plate to a final well volume of 16 μL. For the complex-stabilisation assay, sulfo-Cy5-labelled DCAF16 was titrated 1:4 in TR-FRET assay buffer. Final concentrations of 200 nM for BRD4 constructs and 1 µM for IBG1 were used. Europium anti-His6 donor and DMSO concentrations were kept constant across the plate for both assay formats at 2 nM and 0.5%, respectively. Plates were spun down at 50g for 1 min and covered and incubated at room temperature for 30 min. Afterwards plates were read on the PHERAstar FS.
Instrument settings
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TR-FRET, endpoint
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Optic settings
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Filters
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Filters Ex TR
Em1: 665-10 Em2: 620-10 |
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General settings
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Integration time
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70-400 µs
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| Number of flashes |
200 |
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Settling time
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0 |
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Results & Discussion
A first aim in the presented study was to characterise the possible interactions between DCAF16, BRD4 and the bivalent glue IBG1 in vitro. The formation of a ternary complex between IBG1, DCAF16 and BRD4Tandem, a BRD4 construct containing both bromodomains (BD1 and BD2) connected by the native linker, was observed using isothermal titration calorimetry (ITC; data not shown). Similarly, the time-resolved fluorescence resonance energy transfer (TR-FRET) complex-formation assay showed that a ternary complex formed between DCAF16 and BRD4Tandem in a dose-dependent manner upon IBG1 titration (EC50 = 44 nM; Fig. 2a). Overall, IBG1 was more active than its monovalent precursor JQ1. A complementary TR-FRET-based complex-stabilization assay confi rmed an interaction upon titrating DCAF16 into BRD4Tandem in the presence of IBG1 (Kd = 712 nM; Fig. 2b). Unexpectedly, an intrinsic affinity of DCAF16 to BRD4Tandem in the absence of IBG1 using TR-FRET (Kd = 1 µM; Fig. 2b) was also observed. No such intrinsic affinity was observed with isolated BRD4BD1 or BRD4BD2 indicating that both bromodomains are required for complex formation. Comparison of the ITC titrations for DCAF16 into unbound versus IBG1-bound BRD4Tandem revealed that IBG1 strengthens (Kd of 0.6 µM versus 4 µM; data not shown) and thermodynamically alters the BRD4–DCAF16 interaction.
With the mode of action of IBG1 confirmed, further compounds were synthesised to improve its molecular glue activity towards BRD4 and DCAF16. BRD4 degradation efficiencies of the new compound IBG3 exceeded those of IBG1, with IBG3 showing degradation in a low picomolar range (DC50 = 6.7 pM, data not shown). IBG3 also showed improved ‘gluing’ of the BRD4–DCAF16 complex by TR-FRET (EC50 = 32 nM; Fig. 3).
Similar to its parental compound, IBG3 was specifi c for BRD2 and BRD4 over BRD3, selective for tandem bromodomains over isolated bromodomains, and mediated by DCAF16, indicating degradation via the same intramolecular glue mechanism.
Conclusion
Using the presented TR-FRET-based interaction assay, novel molecular glues targeting the BRD4-DCAF16 interaction could be characterized. These novel intramolecular bivalent glues follow a new interaction mechanism by binding to two domains of the target protein, thereby increasing its affinity to an E3 ligase. This outlines a promising approach to pharmacologically utilize intrinsic interactions with diverse effector proteins and rewire cellular circuits for protein degradation and beyond.
References
- Hsia, O., et al. Targeted protein degradation via intramolecular bivalent glues. Nature 627, 204-211 (2024).