Oct 09, 2024

Why are PPIs so relevant?

Protein-protein interactions (PPIs) play a central role in almost all biological processes, and their dysfunction is often implicated in disease states. Unraveling these complex interactions is essential for understanding basic protein functions. Moreover, PPIs that are frequently affected in disease states have become increasingly relevant as targets for therapeutic discovery.

Various methods have been developed to study and characterize PPIs, including CoIP (co-immunoprecipitation), FRET (Förster resonance energy transfer), X-ray crystallography, ITC (Isothermal Titration Calorimetry), Y2H (yeast two-hybrid assays and more.^1,2,3 Despite these methods, tools to purposefully modulate PPIs to enable their full characterization are not readily available. PPI modulators could also provide new therapeutic opportunities. Therefore, now a team of scientists at VIB-VUB Center for Structural Biology in Belgium, led by Jan Steyaert, has developed a new platform for nanobody generation and selection to facilitate the discovery of PPI modulating tools.

In their recent publication, the team has implemented their approach, which combines Cross-link PPIs and Immunize llamas (ChILL) and Display and co-selection (DISCO) of nanobodies to identify modulators of the SOS1-RAS complex.¹

What are nanobodies?

Nanobodies are single domain-based VHHs or variable antibody fragments normally derived from heavy-chain-only IgG antibodies in Camelids (e.g., Alpacas). Compared to conventional monoclonal antibodies, the much smaller size of VHHs combined with other advantageous properties, such as high binding affinity and stability, has made them highly sought-after tools for various applications.⁴

Unique Properties of Nanobodies.“(A) Diagrammatic representation of the structure of conventional m(IgG)Ab, HCAb, and VHH (Nb), (B) an outline of the unique characteristics found only in Nbs, and (C) an outline of the advantages of Nbs compared to other mAb formats.”⁴ Figure 1 retreived without modifications from Jin et al. 2023.⁴ Deed - Attribution 4.0 International - Creative Commons

Discovering nanobodies to modulate the SOS1-RAS complex

Recombinant nanobodies are well-suited as tools to study and exploit challenging epitopes that may be involved in modulating PPIs. Their compact size enables nanobodies to more easily reach hidden epitopes that otherwise would be inaccessible to conventional monoclonal antibodies.

To favor discovering nanobodies useful in promoting PPIs, Steyaert’s team opted to use a stable protein-protein complex as the immunogen. They reasoned that stabilizing the SOS1-RAS interaction would be critical to expose relevant conformational epitopes, unique to the complex. Therefore, they covalently crosslinked the SOS1-RAS proteins and leveraged the resulting stable complex to immunize llamas.

Immunogen Selection for Nanobody Discovery. Crosslinking of SOS1 and RAS increases the probability of discovering nanobodies specific to conformational epitopes present only upon the formation of a stable PPI complex. Supplementary Figure 1, retrieved without modification from Fischer et al. 2024.¹ Deed - Attribution 4.0 International - Creative Commons

The resulting library of nanobodies generated was subsequently displayed on yeast and screened for binding to fluorescently labeled SOS1-488 and RAS-647 through multicolor fluorescent-activated cell sorting (FACS). This approach enabled the team to distinguish nanobodies based on their binding specificities for each protein and, more importantly, those specific to epitopes present only upon SOS1-RAS complex formation. As such, their approach enabled the identification of nanobodies that could function to disrupt or promote SOS1 and RAS interactions.

Nanobody yeast display screen.. Multicolor fluorescent-activated cell sorting enabled the identification of cells bound to SOS1 (blue), RAS (green), or the SOS1-RAS (blue and green) complex. Figure 1, retrieved without modification from Fischer et al. 2024. Deed - Attribution 4.0 International - Creative Commons

This workflow enabled the team to select nanobodies to further characterize by flow cytometry, Bio-Layer Interferometry (BLI), and X-ray crystallography. Among the nanobodies discovered, Nb77 and Nb84 interacted exclusively with SOS1 and RAS, respectively. Both of these nanobody candidates were effective at inhibiting the GDP/GTP nucleotide exchange by sterically hindering the SOS1-RAS complex formation, as determined through structural analysis.

In contrast, two other nanobodies, Nb14 and Nb22, were found to bind to the SOS1-RAS complex and increase GDP/GTP nucleotide exchange. For instance, Nb14 binds to the complex straddling over SOS1 catalytic and RAS epitopes, stabilizing PPIs and improving activity. Similarly, the binding of a second nanobody, Nb22, which interacts primarily with non-catalytic SOS1 epitopes and binds to a lesser extent to RAS, helps improve catalysis. The team confirmed that the modulation achieved by Nd14 and Nb22 was comparable to that achieved with a natural allosteric modulator, consisting of a RAS mutant (RASY64A) bound to a non-hydrolysable GTP analog.

“Codon-optimized synthetic genes (GenScript) encoding the human KRASG12V and KRASY64A mutants (residues 1–169) and human SOS1 (residues 564–1049) were cloned as NdeI and XhoI fragments into pET28b”¹

With these novel nanobodies in hand, Fischer and colleagues validated their potential to modulate relevant PPIs by focusing on effects downstream of the RAS/MAPK pathway. They found that all nanobodies were effective at reducing ERK1/2 phosphorylation.

Conclusion

Overall, Fischer and colleagues have developed a strong platform for the discovery of nanobodies that modulate relevant PPIs. Their workflow makes it possible to identify PPI-promoting or inhibiting nanobodies, providing tools not only to better understand these interactions but also potentially to the benefit of drug discovery efforts.

Reference

1. Fischer B, et al. (2024). Allosteric nanobodies to study the interactions between SOS1 and RAS. Nature Communications, 15(1), 1-10. https://doi.org/10.1038/s41467-024-50349-2

2. Oláh J, Szénási T, Lehotzky A, Norris V, Ovádi J. (2022). Challenges in Discovering Drugs That Target the Protein-Protein Interactions of Disordered Proteins. Int J Mol Sci. https://doi.org/10.3390%2Fijms23031550

3. Taoka KI, et al. (2021). Novel assays to monitor gene expression and protein-protein interactions in rice using the bioluminescent protein, NanoLuc. Plant Biotechnol (Tokyo). https://doi.org/10.5511%2Fplantbiotechnology.20.1209a

4. Jin BK, Odongo S, Radwanska M, Magez S. (2023). NANOBODIES®: A Review of Diagnostic and Therapeutic Applications. Int J Mol Sci. https://doi.org/10.3390%2Fijms24065994