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

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.

The Why and How of Ultrasmall Nanoparticles

“…52 Another option are thiolated and functionalized macromolecules that efficiently cover the nanoparticle surface. 31,53…”

“… NMR spectroscopy (1D and 2D) enables kind and number of ligands to be determined on the intact nanoparticles (i.e., without detachment of the ligand shell), although the spectra can become crowded and difficult to interpret. , Functionalization is also possible via 1-ethyl-3-(3-dimethylaminopropyl)­carbodiimide (EDC) conjugation of the carboxy group of a tiopronin ligand attached to gold and the amino group of a functional ligand, e.g., doxorubicin or cadaverine . Another option are thiolated and functionalized macromolecules that efficiently cover the nanoparticle surface. , …”

Kinetics and Timescales in Bio–Nano Interactions

“…Figure 4B, in particular, clearly illustrates the distinct ranges of k off and t r that are characteristic of ultrasmall versus conventional NPs. Of note, one should bear in mind that certain types of usNPs may exhibit enhanced binding stability and longer residence times, including functional usNPs [107][108][109][110], dendrimers with exceptionally high charge densities, or hydrophobic carbon NPs like fullerenes.…”

Multivalent Calixarene Complexation of a Designed Pentameric Lectin

“…6−10 One approach to glyco-clustering for lectin complexation relies on calix[n]arene scaffolds. 11−15 In the case of calix [4]arene, variants bearing one to four arms with carbohydrate hands have been used to complex lectins with up to five binding sites. 12,13 A penta-glycosylated calix [5]arene was found to have 10 5 -fold tighter binding to Cholera toxin than the monomeric ganglioside GM1 oligosaccharide.…”

“…Multivalency is central to biological interactions from bimolecular events such as protein–ligand complexation to multimolecular processes such as agglutination and cell–cell recognition. − Here, we describe multivalent host–guest complexation between a designed pentameric lectin and the synthetic octameric macrocycle sulfonato-calix[8]­arene ( sclx 8 ). The mechanism of multivalency contrasts with that employed in previous lectin-binding studies.…”