Detection technologies to study protein-protein interactions

Dr Barry Whyte discusses how microplate readers could help accelerate drug discovery and development.

Protein-protein interactions, which underpin many crucial molecular events taking place in a cell, are an important area of research and discovery. A typical cell contains thousands of proteins and few of them function alone. It is therefore vital to study how proteins interact with one another and the tools to investigate these interactions have continued to advance over time. Applications amenable to scale can significantly accelerate research and discovery of inhibitors and drugs, for instance. Here, we look at a few examples of how detection technologies in microplate readers provide options for the study of protein-protein interactions in the rapidly emerging area of targeted protein degradation.

Different options to study protein-protein interactions

Microplate readers support many useful applications in the life sciences due to their ability to provide ready access to a range of detection technologies. Protein-protein interactions require efficient, highly-sensitive assays often with many measurements in a short period of time. For the screening of molecules, it is crucial to have detection technologies that are compatible with automation, and which deliver the required sensitivity for miniaturised assays. Fluorescence- and luminescence-based measurements are often used for this purpose. In addition to detection methods based on Förster’s Resonance Energy Transfer (FRET), fluorescence polarisation is an advanced detection mode that offers exciting opportunities for the study of protein-protein interactions.

ATTECs (Autophagy-Tethering Compounds) are a novel class of bifunctional molecules proposed to hijack the autophagosomal pathway for the degradation of cellular components including proteins. The induction of Atg8 family protein interactions with target proteins of interest offers the possibility for novel targeted protein degradation approaches. LC3A is a member of the human Atg8 protein family and is only found in the autophagosome. The discovery of potent LC3A inhibitors would therefore serve as a handle for the development of ATTECs.1

In the following example, a fluorescence polarisation assay was developed based on the use of a fluorescently labelled peptide ligand that binds to LC3A. p62 LIR peptide, a peptide ligand of LC3A, was labelled with a Cy5 fluorophore to act as a tracer molecule. The addition of competing ligands of LC3A to the LC3A-tracer complex results in a displacement of tracer and a reduction of the fluorescence polarisation signal (Figure 1). Miniaturisation of this assay allowed for library screening in 1536-well plates (Figure 2) that was suitable with automation for screening of large compound libraries in days.

In another example related to targeted protein degradation, a NanoBRET-based approach was used to look at the dose-dependent ubiquitination of BRD4. Bromodomain-containing protein 4 is a transcriptional regulator implicated in cancer biology and inflammation. Ubiquitination is a crucial step in the action of PROTACs, small molecules that help target unwanted proteins to the ubiquitin-proteasome system. In this case, PROTAC ARV-771 brought a BRD4-labeled HiBiT (a subunit of the NanoLuc luciferase) into proximity with the E3 ubiquitin ligase. Ubiquitin tags on the target protein earmarked it for degradation by the proteasome. Here it was possible to measure the live cell kinetics of ternary complex formation with the E3 ligase as well as the efficiency with which the target protein is ubiquitinated, crucial information to confirm mode of action and to probe ways to improve drug efficacy (Figure 3).

What microplate readers bring to studies of protein-protein interactions

Many applications in the laboratory need to be performed at scale and sensitivity and speed are crucial. High-throughput screening assays need to be compatible with automation to accelerate measurements but must also deliver the required sensitivity.

Binding studies for protein-protein interactions provide valuable information on reaction kinetics, dissociation constants and the stoichiometry of interactions. FRET, for example, can be used together with dye-labelled proteins to determine the stoichiometry of protein interactions. Awareness of stoichiometry can be crucial for example in calculations to quantify labelled biomolecules that assemble or are active in different ratios (dimers, trimers, etc.). Studies on binding kinetics on BMG Labtech microplate readers are facilitated by the MARS data analysis software.

Multimode microplate readers like the PHERAstar FSX from BMG Labtech offer many features that make them ideal platforms for research applications at scale (Figure 4). Users have flexibility of choice since all commonly-used detection modes are available at the performance level required for screening of molecules. In addition, features such as on-board reagent injectors, simultaneous dual emission for the detection of two emission signals at the same time, and ultrafast sampling rates with detection times of up to 0.01s allow kinetic analysis of interactions in real time at high throughput. Collectively, these features provide the robust high performance needed for automated applications at scale in the modern research laboratory.

 

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