Collateral-ID: Mapping the off-target degradome of PROTACs

Essebtial background: Drug discovery has traditionally focused on the development of small molecules that act by binding and inhibiting the enzymatic activity of proteins. Due to their mechanism of action, traditional small molecule drugs are not suited to target a large proportion of the proteome. Targeted protein degradation (TPD) is a new drug modality that can be used to eliminate disease-causing proteins that have been deemed undruggable in the past 1.

Due to its potential, it is one of the fastest area of growth in biopharma, with the first class of molecules in clinical trial2. Proteolysis-targeting chimeras (PROTACs) are a type of TPD molecule that works with the ubiquitin-proteasome system to degrade proteins. Protein degradation has the advantage of removing all functions of the target (i.e. structural, regulatory, enzymatic etc.) allowing proteins without enzymatic activity to be therapeutically targeted.

Moreover, design is not constrained by a requirement of strong binding and disruption of enzymatic activity as PROTACs only require transient interaction with any surface of a protein to promote degradation. PROTACs therefore have the potential to significantly increase the number of targets that can be therapeutically engaged.

PROTACs are heterobifunctional molecules compose of E3 ligase and protein of interest (POI) ligands connected by a chemical linker. When the E3-PROTAC-POI complex (ternary complex) is formed, it leads to the ubiquitination and degradation of the POI. These ternary complexes are artificial, and their formation can have several unintended consequences within the cells.

One consequence unique to this modality is binding and potential degradation of off-target proteins that interact with the PROTAC and/or E3-PROTAC-POI complex (Figure 1A)3.

Routine in vitro pharmacology profiling, which measures small molecule binding to a broad panel of targets implicated in adverse drug events are unable to test many of these potential off-target effects of PROTACs. While efforts are being made to develop whole-cell mass spectrometry-based approaches to measure off-target protein degradation 4, currently they lack the sensitivity required to measure changes in low-abundance proteins.

This project will establish a platform using protein proximity labelling to interrogate the protein complexes formed by PROTACs: Collateral-ID. Protein proximity labelling utilises a biotin ligase that in the presence of excess biotin will transfer biotin to lysines on nearby proteins. By fusing the biotin ligase to a protein of interest (bait protein), one can specifically label interacting proteins.

The system allows for weak and transient interactions to be efficiently detected. These biotinylated proteins can be purified by streptavidin pull down and identified via mass spectrometry. In the case of Collateral-ID the bait protein will be the E3 ligase being utilised by the PROTAC under investigation.

Proteins that are proximal to the E3 ligase can be identified with and without PROTAC being present. Importantly, these experiments will be carried out under proteasome inhibition to prevent proteasomal degradation of biotin-tagged proteins that would otherwise occur due to ubiquitination mediated by the E3 ligase (Figure 1B). Following identification of proteins that are in complex with E3 ligase we can assess whether these proteins are degraded as a result of PROTAC treatment using methods such as western blotting and immunofluorescence or utilise live-cell imaging of fluorescently tagged protein to understand degradation kinetics.

Importantly, proximity labelling has already been successfully used to interrogate the dynamics of PROTAC induced complexes 5 6. This targeted approach will provide a much-needed tool to probe off-target effects and inform POI and E3 selection for PROTAC technology development.