BRD4 PROTAC Characterization using SPR

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Our bioscience team undertook a project on developing a Surface Plasmon Resonance (SPR) assay to enable kinetic and affinity measurements of well-known BRD4-targeting PROTAC molecules. Collecting data that allows us to infer different mechanistic details of the PROTAC / BRD4 interactions. 

We have previously combined a fluorescence-based thermal shift assay (FTSA) and cell-based protein degradation experiments (using our ‘Jess’ semi-automated Western blotting system) to demonstrate the binding and efficacy of several BRD4-targeting PROTAC molecules. 

In this project we developed an SPR assay to measure the kinetics and binding affinity of several literature-derived PROTAC and small molecule compounds to the well-known exemplar protein ‘BRD4’. Through this we could demonstrate insights into subtle differences in binding mode between BRD4 and seemingly very similar PROTAC molecules – something that can be applied to future SPR-based PROTAC screening projects.

Using amine-coupling chemistry we could immobilize BRD4 readily to the surface of a CM5 chip and this generated a very robust surface for binding experiments. We measured the binding to BRD4 of drug-like small molecules and much larger PROTAC molecules, measuring kinetic and affinity data for both groups.

The small molecule affinities closely matched reported affinity/potency values from literature, validating our assay.

For the two PROTAC molecules we saw subtle differences in binding mode, hinted at by the deviation away from a good fit to the 1:1 binding model for one of these. The only difference between the two PROTAC molecules was the linker region and the ligase-targeting warhead – neither of which would be expected to influence the PROTACs’ binding to BRD4. Indeed. Both compounds shared the same BRD4-targeting warhead.

You can see further explanation of our findings below.

BP-Immobilisation-Figure

Figure 1. Typical workflow for determining amine coupling conditions for a new protein. Top left -pH Scouting identifies the best condition for the ‘pre-concentration’ stage. Bottom left, bottom right –  We had to drastically reduce the protein contact time to ensure we did not immobilize too much BRD4. Top right – We typically observe a baseline post-immobilization to ensure that it stabilizes for compound binding work. 

BP-JQ1-Figure

Figure 2.  JQ1(+) is the BRD4-targeting warhead on both of the PROTAC compounds described above. We see here excellent quality sensorgrams that allow us to confidently measure the kinetics and affinity of the binding between JQ1(+) and BRD4.

BP-PROTAC-Figure

Figure 3. We measured the binding of two PROTACs against our BRD4 surface. Both PROTACs share the same BRD4-targeting warhead but differ in their linker region and E3 ligase-targeting warhead. These differences apparently affect their respective binding to BRD4 – in the case of dBET6 we see an excellent fit from the 1:1 binding model whereas from ARV-771 this is not the case.

These results demonstrate how SPR can provide interesting and important characterization of binding modes that might be assumed to be very similar – in PROTAC projects or beyond.

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