Announcement: Aurelia Bioscience has been acquired by Charnwood Molecular.

Share this

The poster titled Beyond Degradation: Cell-based and Biophysical PROTAC Characterisation in a CRO outlines how Charnwood Molecular used cell-based and biophysical techniques to investigate - amongst others - the BRD4-targeting PROTAC 'dBET6' to elucidate a binding mechanism, confirm proteasomal degradation and monitor its impact on cellular behaviour.

Read the full abstract here: 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 cycle. A number of PROTACs have previously been developed to target the well-studied protein BRD4 - a member of the BET (bromodomain and extra-terminal) family of proteins whose dysregulation is linked to human cancers.

We have used cell-based and biophysical techniques to investigate - amongst others - the BRD4-targeting PROTAC 'dBET6' to elucidate a binding mechanism, confirm proteasomal degradation and monitor its impact on cellular behaviour. Here we demonstrate the techniques that we might typically implement in a PROTAC development cascade.

Biophysics approaches such as SPR can be used to measure the binding kinetics and affinity of warhead or whole PROTAC molecules to their target whilst cell-based assays focus on examining the behaviour of the compound in their intended environment. SPR was performed using the Biacore 8K to assess differences in how dBET6 and the BDR4-targeting warhead 'JQ1(+)' (from which dBET6 was derived) interact with immobilised BRD4 protein.

The JESS automated immunoblotting platform is an ideal tool to quantify target protein levels in complex cellular samples and here we use it to confirm PROTAC-mediated degradation of BRD4 and measure the levels of proteins downstream of BRD4-regulated expression.

Live-cell imaging using our IncuCyte SX5 instrument complements this by providing insights into phenotypic changes at the cellular level. In this instance migration of cells in a scratch wound model was investigated plus the ability of dBET6 to induce apoptosis in the same cell line.

This set of orthogonal assays was used to probe BRD4 PROTAC activity, but they could be equally utilised in the development of PROTACs for a wide range of other targets alongside other more target-specific bespoke assays.

At Charnwood Molecular we have applied our extensive experience and our integrated approach to drug discovery to a number of different PROTAC-development projects on behalf of our clients.

Download the poster here.

Find out more about SPR and relevant case studies.