Potentiating Tweezer Affinity to a Protein Interface with Sequence-Defined Macromolecules on Nanoparticles

Survivin,a well-known member of the inhibitor of apoptosis proteinfamily, is upregulated in many cancer cells, which is associated withresistance to chemotherapy. To circumvent this, inhibitors are currentlybeing developed to interfere with the nuclear export of survivin bytargeting its protein-protein interaction (PPI) with the exportreceptor CRM1. Here, we combine for the first time a supramoleculartweezer motif, sequence-defined macromolecular scaffolds, and ultrasmallAu nanoparticles (us-AuNPs) to tailor a high avidity inhibitor targetingthe survivin-CRM1 interaction. A series of biophysical and biochemicalexperiments, including surface plasmon resonance measurements andtheir multivalent evaluation by EVILFIT, reveal that for divalentmacromolecular constructs with increasing linker distance, the longestlinkers show superior affinity, slower dissociation, as well as moreefficient PPI inhibition. As a drawback, these macromolecular tweezerconjugates do not enter cells, a critical feature for potential applications.The problem is solved by immobilizing the tweezer conjugates ontous-AuNPs, which enables efficient transport into HeLa cells. On thenanoparticles, the tweezer valency rises from 2 to 16 and producesa 100-fold avidity increase. The hierarchical combination of differentscaffolds and controlled multivalent presentation of supramolecularbinders was the key to the development of highly efficient survivin-CRM1competitors. This concept may also be useful for other PPIs.