Protein-protein interactions (PPIs) lie at the heart of biology. Here we explain how Depixus MAGNA One™ can be used to analyze dynamic protein-protein interactions at scale, providing deep insights into biology and accelerating the development of novel therapeutics such as PPI inhibitors and molecular glues.

What are protein-protein interactions and why are they important?

Protein-protein interactions (PPIs) govern nearly every cellular process, from immune responses to cell signaling and metabolic regulation.

These interactions are known collectively as the interactome – the complex network of interactions within cells and organisms. The emerging field of interactomics focuses on mapping and understanding these interactions, providing crucial insights into cellular biology and disease mechanisms.

From oncology to rare diseases and neurological conditions, targeting PPIs holds enormous therapeutic promise, offering opportunities to intervene in critical biological pathways across numerous indications.

However, PPIs remain elusive as drug targets and were, until recently, considered undruggable and hard to study due to their highly dynamic and transient nature.¹

In this blog, we will explore the current status of PPI-targeting drugs and the ways in which scientists are studying these interactions to drive forward the development of novel therapeutics.

Unlocking the therapeutic potential of protein-protein interactions

Researchers can disrupt or restore crucial biological pathways by inhibiting, stabilizing or inducing specific protein-protein interactions. Diverse modalities, from small molecules to antibodies and aptamers, can be employed to disrupt harmful PPIs or restore beneficial ones.²

For example, Venetoclax (ABT-199) is a small molecule that prevents the interaction between Bcl-2, a pro-survival protein, and Bax, a key regulator of apoptosis in cancer cells. Approved by the FDA in 2016, Venetoclax is used to treat relapsed or recurrent acute myeloid leukemia through inhibition of this specific PPI.³

Another example is Sirolimus (rapamycin), an effective immunosuppressant often used in organ transplantation and to treat autoimmune diseases. It works by stabilizing a complex between the proteins FKBP12 and mTORC1, inhibiting a critical pathway in T-cell proliferation that is an essential part of mounting an effective immune response.⁴

Molecular glues and PROTACs: Novel therapeutic approaches targeting protein-protein interactions

Proximity-inducing therapeutics such as molecular glues and bifunctional molecules have expanded the range of protein targets to include many previously considered to be undruggable.

A prominent area of study is targeted protein degradation, where PROTACs (proteolysis-targeting chimeras) and molecular glues play key roles. Molecular glues are compounds that stabilize or induce new protein-protein interactions, with one subset – the degraders – utilized for targeted protein degradation.²

Both PROTACs and molecular glues induce an interaction between a target protein and an E3 ligase complex to promote targeted protein degradation via the ubiquitination pathway. Such targeted protein degradation is already transforming drug development and opening new possibilities in treating complex diseases.^2,5

As an example, the multiple myeloma drug Revlimid (lenalidomide) induces a novel interaction between the protein CRBN of the E3 ligase complex CRL4 ^CRBN and the transcription factors IKZF1/3, which are essential for the survival of malignant B cells. This promotes ubiquitination of IKZF1/3 and their subsequent targeting for proteasomal degradation.⁶

These success stories demonstrate the transformative potential of targeting PPIs to treat disease. However, the pace of novel PPI therapeutics coming to the clinic has been slow, due to the challenges of finding viable drugs targets within relatively featureless protein surfaces and the inherent technical challenges of studying these dynamic and transient interactions.

Limitations of current methods for studying protein-protein interactions

Scientists rely on a diverse array of experimental methods to develop therapies targeting PPIs, each uncovering unique aspects of these interactions.¹

• Biochemical techniques: Phage display and affinity purification coupled with mass spectrometry (AP-MS) are widely used to identify PPIs.
• Cell-based assays: Techniques such as Förster resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC)⁷ allow researchers to study PPIs in fixed or living cells.
• Biophysical methods: Surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and microscale thermophoresis (MST) provide insights into the kinetics and thermodynamics of protein interactions.
• Single-molecule technologies: Tools like optical tweezers, atomic force microscopy (AFM), and single-molecule FRET (smFRET) capture rare events and heterogeneity in PPI dynamics.
• Structural techniques: High-resolution methods such as X-ray crystallography, nuclear magnetic resonance (NMR), and cryo-electron microscopy (cryo-EM) reveal atomic-level details.

To overcome the limitations of individual techniques, researchers often rely on a combination of methods to gain a comprehensive understanding of protein-protein interactions.

However, obtaining a full picture of PPI kinetics, energetics, and heterogeneity remains time-intensive, technically complex, and laborious.¹ Here’s why:

• Specialization: Techniques like cryo-EM and NMR, along with most single-molecule technologies, demand extensive expertise and only allow examination of a small number of molecules, making it hard to capture heterogeneity.
• Sample requirements: Some approaches, such as ITC, require large quantities of experimental material, posing challenges for hard-to-purify proteins.
• Transient interactions: Many methods struggle to detect fast, transient, or weak PPIs, which are often biologically significant.
• Rare events: Bulk techniques generate averaged results and often miss rare but biologically significant interactions.

Depixus MAGNA One addresses these limitations, offering a faster, simpler and more accessible alternative for studying thousands of individual dynamic protein-protein interactions in parallel.

Depixus MAGNA One™ analyzes dynamic protein-protein interactions at scale

Based on magnetic force spectroscopy, Depixus MAGNA One enables you to directly measure thousands of dynamic PPIs in real-time without the need for large quantities of starting material. And with high precision and sensitivity, you can detect even weak or transient interactions that traditional methods miss.

Figure 1 below shows a PPI being measured using Depixus MAGNA One. Two interacting proteins (blue and pink) are tethered to a nucleotide scaffold that is attached to a surface at one end and a paramagnetic bead at the other. A highly controllable magnet is then used to apply a force to the beads, while a visual tracking system provides precise data on the vertical position of each bead.

 

Figure 1. Schematic showing how protein-protein interactions can be measured using Depixus MAGNA One.

When little or no magnetic force is applied, the tethered proteins can interact and separate freely (Figure 1, left). The bead vertical position allows these subtle movements to be monitored in real time, providing valuable information about the kinetics of PPIs. This data is particularly useful for studying weak and intermittent PPIs, which may be challenging to capture using other techniques.

Slowly increasing the magnetic force causes the beads to rise, extending the scaffold (Figure 1, middle). The interacting proteins then cause a pause in the extension until, at a given force, the PPI is overcome and the two proteins separate. The bead then rises to the maximum height allowed by the scaffold (Figure 1, right).

Tracking the position of each bead provides detailed data about the force required to disrupt the PPI (Figure 2). Ligands such as small molecules can then be introduced to allow their effect on the strength and stability of the interaction to be studied.

 

Figure 2. Example traces of bead vertical position as the force is increased for a pair of proteins that are interacting (blue trace) and not interacting (orange trace). The protein-protein interaction prevents the bead from fully rising until the applied force is sufficient to separate the proteins, resulting in a sharp change in bead position.

This process is scaled up over thousands of molecules in parallel within the Depixus MAGNA One instrument, providing a uniquely rich data stream.

The detailed dynamic data about PPIs that can be generated at scale by Depixus MAGNA One has the potential to significantly accelerate the development of novel therapeutics and unlock huge value in this exciting area.

Ready to learn more? To explore in detail how Depixus MAGNA One™ can be used to visualize protein-protein interactions, uncover the dynamics and binding strength of ternary complexes, and study the effectiveness of molecular glues download our application note today.

References

1. Hayes S, et al. Studying protein–protein interactions: progress, pitfalls and solutions. Biochemical Society Transactions. 2016 Aug 15;44(4):994–1004. doi: 10.1042/bst20160092

2. Konstantinidou M, Arkin MR. Molecular glues for protein-protein interactions: Progressing toward a new dream. Cell Chem Biol. 2024 Jun 20;31(6):1064-1088. doi: 10.1016/j.chembiol.2024.04.002

3. Souers AJ, et al. ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets. Nat Med. 2013 Feb;19(2):202-8. doi: 10.1038/nm.3048

4. Li J, Kim SG, Blenis J. Rapamycin: one drug, many effects. Cell Metab. 2014 Mar 4;19(3):373-9. doi: 10.1016/j.cmet.2014.01.001

5. An S, Fu L. Small-molecule PROTACs: An emerging and promising approach for the development of targeted therapy drugs. EBioMedicine. 2018 Oct;36:553-562. doi: 10.1016/j.ebiom.2018.09.005

6. Kulig P, et al. Lenalidomide in Multiple Myeloma: Review of Resistance Mechanisms, Current Treatment Strategies and Future Perspectives. Cancers (Basel). 2023 Feb 2;15(3):963. doi: 10.3390/cancers15030963

7. Ren H, et al. Comprehensive Review on Bimolecular Fluorescence Complementation and Its Application in Deciphering Protein-Protein Interactions in Cell Signaling Pathways. Biomolecules. 2024 Jul 17;14(7):859. doi: 10.3390/biom14070859