Related Experiment Video
Updated: Jul 21, 2025
Monitoring Conformational Dynamics of Single Unmodified Proteins using Plasmonic Nanotweezers
Published on: March 21, 2025
Potentiating Tweezer Affinity to a Protein Interface with Sequence-Defined Macromolecules on Nanoparticles
Theresa Seiler1, Annika Lennartz2, Kai Klein3
1Department for Organic Chemistry and Macromolecular Chemistry, Heinrich Heine University Duesseldorf, Universitaetsstraße 1, Duesseldorf 40225, Germany.
Abstract:
Survivin, a well-known member of the inhibitor of apoptosis protein family, is upregulated in many cancer cells, which is associated with resistance to chemotherapy. To circumvent this, inhibitors are currently being developed to interfere with the nuclear export of survivin by targeting its protein-protein interaction (PPI) with the export receptor CRM1. Here, we combine for the first time a supramolecular tweezer motif, sequence-defined macromolecular scaffolds, and ultrasmall Au nanoparticles (us-AuNPs) to tailor a high avidity inhibitor targeting the survivin-CRM1 interaction. A series of biophysical and biochemical experiments, including surface plasmon resonance measurements and their multivalent evaluation by EVILFIT, reveal that for divalent macromolecular constructs with increasing linker distance, the longest linkers show superior affinity, slower dissociation, as well as more efficient PPI inhibition. As a drawback, these macromolecular tweezer conjugates do not enter cells, a critical feature for potential applications. The problem is solved by immobilizing the tweezer conjugates onto us-AuNPs, which enables efficient transport into HeLa cells. On the nanoparticles, the tweezer valency rises from 2 to 16 and produces a 100-fold avidity increase. The hierarchical combination of different scaffolds and controlled multivalent presentation of supramolecular binders was the key to the development of highly efficient survivin-CRM1 competitors. This concept may also be useful for other PPIs.
Insights
Researchers developed novel inhibitors targeting cancer-promoting survivin by blocking its interaction with CRM1. Immobilizing these inhibitors on nanoparticles enhanced cellular uptake and significantly boosted their effectiveness against cancer cells.
Area of Science:
- Biochemistry
- Nanotechnology
- Molecular Biology
Background:
- Survivin, an apoptosis inhibitor, is upregulated in cancers, contributing to chemotherapy resistance.
- Inhibiting survivin's interaction with the CRM1 export receptor is a strategy to overcome resistance.
- Targeting protein-protein interactions (PPIs) is crucial for developing novel cancer therapies.
Purpose of the Study:
- To design and synthesize a novel high avidity inhibitor targeting the survivin-CRM1 interaction.
- To investigate the impact of multivalent presentation and nanoparticle conjugation on inhibitor efficacy.
- To demonstrate efficient cellular delivery and enhanced inhibition of survivin-CRM1 PPI.
Main Methods:
- Utilized supramolecular tweezer motifs, sequence-defined macromolecular scaffolds, and ultrasmall gold nanoparticles (us-AuNPs).
- Employed biophysical techniques like surface plasmon resonance (SPR) and computational analysis (EVILFIT) for affinity and avidity measurements.
- Conjugated tweezer inhibitors to us-AuNPs for enhanced cellular uptake and evaluated their inhibitory effects in HeLa cells.
Main Results:
- Macromolecular tweezer constructs with longer linkers exhibited superior affinity and slower dissociation rates.
- Macromolecular tweezer conjugates alone did not enter cells.
- Immobilization on us-AuNPs enabled efficient cellular transport and increased inhibitor avidity by 100-fold due to increased valency (up to 16).
Conclusions:
- A hierarchical approach combining supramolecular binders, scaffolds, and nanoparticles effectively creates potent inhibitors of the survivin-CRM1 PPI.
- Nanoparticle-mediated delivery overcomes cellular entry limitations of macromolecular inhibitors.
- This strategy offers a promising platform for developing inhibitors against other critical protein-protein interactions in cancer therapy.
