Beyond Degradation: Cell Based and Biophysical Techniques of PROTAC Characterization

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During the development of PROTAC compounds to degrade Bromodomain protein 4 (BRD4) specifically for cancer therapy, we have to think about several different things. 

Challenges

BRD4 is a transcriptional and epigenetic regulator that plays a pivotal role in cancer and inflammatory diseases. It binds and remains associated with chromatin during mitosis, bookmarking early G1 genes and reactivating transcription after mitotic silencing. BRD4 plays an important role in transcription, both as a passive scaffold via its recruitment of vital transcription factors and as an active kinase that phosphorylates RNA polymerase II, directly and indirectly regulating transcription. 

As an integrated project working with our chemistry and ADME/DMPK colleagues, Charnwood Discovery developed specific PROTAC molecules for BRD4 degradation through the proteosome system.

PROTAC’s are bivalent molecules composed of two binding molecules or warheads, one binds specifically to the target protein, in this case BRD4, the second that binds the ligase, generally either Cereblon (CRBN) or Von Hippel Ligase (VHL). 

The physiological role of ligases are to add ubiquitin to lysine groups on proteins and, by doing so, targeting the protein for destruction by the proteosome. Both warheads are joined by a chemical linker of appropriate length, to allow close proximity of the target protein with the ligase such that ternary complex formation can occur.

Targeted protein degradation

Figure 1. A PROTAC molecule interacting with the protein of interest (POI) and E3 ligase bring them into close proximity.

In the development of PROTACs it is vital to have a varied suite of assays that can inform the structure-activity relationship (SAR) and progress the design-make-test-analyse cycle. At Charnwood Discovery, we have used cell-based assays, biophysical techniques and phenotypic approaches to investigate BRD4-targeting PROTAC’s to elucidate the binding mechanism, confirm proteasomal degradation and monitor its impact on cellular behaviour.

What We Did...

Characterizing Binding Modes Using SPR

SPR is a well-regarded biophysical technique that can be applied equally to initial primary screening of compound libraries as well as detailed characterisation of an individual molecular interaction. The data below shows characterisation performed on our Biacore 8K instrument, measuring the kinetics and affinity of a PROTAC molecule dBET6 and the warhead JQ1(+) against BRD4 protein.

Beyond Degradation

Figure 2. Detection of binding of JQ1(+) to BRD4 protein in the Biacore 8K instrument.

In the case of the BRD4-targeting warhead JQ1(+), we can measure very high quality sensorgrams for its interaction with BRD4. At some of the higher concentrations of compound, we observe a deviation away from 1:1 binding, and these sensorgrams have been excluded to allow a good fit of the model to the data.

The subsequent kinetic fit (KD = 26nM) shows good agreement with the affinity fit (KD = 24nM).

The subsequent kinetic fit (KD = 26nM) shows good agreement with the affinity fit (KD = 24nM).

Figure 3. Kinetic fit plot of JQ1(+) to BRD4 protein.

PROTAC molecule dBET6 is based on JQ1(+) – using JQ1(+) as its BRD4-targeting warhead. In similar binding experiments, dBET6 shows an excellent fit to a 1:1 binding model at all concentrations – presumably some properties conferred to the PROTAC molecule from either the linker region or the cereblon-targeting warhead have positively influenced the molecule’s binding to BRD4 and general solubility-related properties.

Beyond Degradation

Figure 4. Detection of binding of dBET6 to BRD4 protein in the Biacore 8K instrument.

The subsequent kinetic fit (KD = 4.4nM) shows excellent agreement with the affinity fit (KD = 4.1nM).

Beyond Degradation

Figure 5. Kinetic fit plot of dBET6 binding to BRD4 protein

Monitoring PROTAC-Inducuded Protein Degradation

Confirmation of target protein degradation is an essential element of any PROTAC development cascade. The data in Figure 6 was generated using the JESS™, a Simple Western™ automated western blot system, to investigate BRD4 degradation induced by dBET6, a commercially available PROTAC degrader of BRD4.

Figure 6. The pseudo blot image below visualized BRD4 and actin (used as a loading control) in MCF-7 cell lysates produced after 24 hours of culture in the presence of a range of dBET6 concentrations. BRD4 was visualized in the chemiluminescence channel together with actin as a loading control in the near infra-red channel.

Figure 7. BRD4 protein levels in MCF-7 cells following dBET6 treatment. Western blot BRD4 bands were normalised to actin as a loading control.

Treatment with dBET6 results in a concentration-dependent reduction in BRD4 signal, with an DC50 of 14nM. To confirm proteasomal degradation, the proteosome inhibitor MG132 (used at 1 µM) was dosed together with 100nM dBET6 and incubated over a 24-hour period. This successfully inhibited degradation of BRD4 but did not impact BRD4 levels in the vehicle treated DMSO controls.

Beyond Degradation

Figure 8. Inhibition of proteosome prevents degradation of BRD4, confirming a PROTAC degradation mechanism for dBET6

Beyond Degradation

Figure 9. Recovery of BRD4 protein in MCF7 cells 24hrs after removal of dBET6 and subsequent replacement of media. At the 24-hour recovery period a very small increase in dBET6 protein was detected.

We also performed a washout experiment to examine the timeframe of BRD4 recovery after 24 hours incubation with the PROTAC. Extracellular dBET6 was removed from MCF-7 cultures and lysates produced 2, 4, 6 and 24 hours post-washout. Even 24 hours after dBET6 was removed from in vitro cultures, BRD4 levels had not recovered to the levels observed in vehicle-treated controls.

As BRD4 is involved in regulating the transcription of several genes (c-Myc and Aurora kinase B for example), the downstream impacts of degradation was also investigated using qPCR to monitor mRNA levels, or to investigate these targets at the protein level using automated immunoblotting.

Phenotypic Assays: Scratch Wound Migration and Cell Health

We have used the IncuCyte® SX5 live-cell kinetic imager to monitor cell behaviour in real-time and understand the phenotypic responses to PROTAC treatment. Here, we use a scratch wound model to investigate the ability of MCF-7 cells to migrate when treated with serial dilutions of compounds. Cytochalasin D, an actin polymerisation inhibitor, was used as a positive control to compare to dBET6 and JQ1(+).

Beyond Degradation_Cell Based and Biophysical Techniques of PROTAC Characterization_9

Figure 10. Scratch wound assay. Image analysis was handled by trainable algorithms that identified the initial scratch wound and tracked wound closure over the course of the experiment to calculate a “relative wound density”.

Cell infill was observed over 72 hours and area under curve (AUC) analysis used to plot kinetic data (below), showing that dBET6, but not JQ1(+), inhibited cell migration into the scratch in a concentration-dependent manner. dBET6 was a more potent inhibitor of migration in MCF-7 cells than cytochalasin D (producing IC50’s of 83nM and 391nM respectively) but did not fully inhibit migration at high concentrations. 

Figure 11. Relative wound density over 72 hours following exposure of MCF7 cells to dBET6. A dose-dependent decrease in wound closure was observed.

Figure 12. JQ1(+), the ‘warhead’ of dBET6, did not inhibit wound closure whereas dBET6, the PROTAC containing the JQ1(+) warhead, did inhibit cellular infill into the scratch area. Cytochalasin D, the positive control, also inhibited cellular infill through a different mechanism.

Kinetic imaging was also used to monitor cell health in the presence of BRD4-interacting compounds. MCF-7 confluency measured over 72 hours shows that dBET6 inhibited proliferation with an IC50 of 54nM, compared to JQ1(+) at 3 µM.

Figure 13. Both dBET6 and JQ1(+) were able to inhibit the growth of MCF7 cells, however dBET6 inhibits cell division and growth more significantly with greater potency than the targeting warhead JQ1(+) alone.

Figure 14. In MCF7 cells, dBET6 was able to induce caspase 3/7 activation more potently than the warhead JQ1(+).

Summary

Apoptosis induction was detected using a cell-permeable dye that produces a signal on caspase 3/7 activation. This also showed that dBET6 (EC50 71nM) is a more potent inducer of apoptosis than JQ1(+). These results highlight the difference in phenotypic response in target protein degradation (dBET6) compared to inhibition (JQ1(+). This phenomenon of PROTAC molecules exhibiting greater potency, efficacy, and selectivity than their targeting protein warhead has been observed in other studies in the literature and at Charnwood Discovery.