Abstract
Here we exploit the simple, ultra-stable, modular architecture of consensus-designed tetratricopeptide repeat proteins (CTPRs) to create a platform capable of displaying both single as well as multiple functions and with diverse programmable geometrical arrangements by grafting non-helical short linear binding motifs (SLiMs) onto the loops between adjacent repeats. As proof of concept, we built synthetic CTPRs to bind and inhibit the human tankyrase proteins (hTNKS), which play a key role in Wnt signaling and are upregulated in cancer. A series of mono-valent and multi-valent hTNKS binders was assembled. To fully exploit the modular scaffold and to further diversify the multi-valent geometry, we engineered the binding modules with two different formats, one monomeric and the other trimeric. We show that the designed proteins are stable, correctly folded and capable of binding to and inhibiting the cellular activity of hTNKS leading to downregulation of the Wnt pathway. Multivalency in both the CTPR protein arrays and the hTNKS target results in the formation of large macromolecular assemblies, which can be visualized both in vitro and in the cell. When delivered into the cell by nanoparticle encapsulation, the multivalent CTPR proteins displayed exceptional activity. They are able to inhibit Wnt signaling where small molecule inhibitors have failed to date. Our results point to the tremendous potential of the CTPR platform to exploit a range of SLiMs and assemble synthetic binding molecules with built-in multivalent capabilities and precise, pre-programmed geometries.
Highlights
The relationship between protein structure and function has been a cornerstone of biology for decades
We functionalised the consensus-designed tetratricopeptide repeat proteins (CTPRs) scaffold by gra ing an 8-residue human tankyrase proteins (hTNKS)-binding consensus peptide (TBP), REAGDGEE, identi ed from a mutational analysis of hTNKS substrates,[36] onto the loop between two adjacent repeats (Fig. 1A)
We created potent hTNKS inhibitors by combining target speci city with multivalency, two features that have not been explored in previous drug development efforts against this target
Summary
The relationship between protein structure and function has been a cornerstone of biology for decades. In recent years, the unstructured or intrinsically disordered regions of the eukaryotic proteome (40% in humans) have gained increasing interest This is due in part to the abundance in these regions of short independently functioning binding modules known as MoRFs (molecular recognition features) or SLiMs (short linear motifs).[1] One approach to exploit these motifs for inhibiting protein–protein interactions (PPIs) is to chemically synthesise them in combination with modi cations such as cross-linking and macrocyclisation designed to improve affinity, half-life and cell penetration.[2] in nature high-affinity and high-speci city interactions and more complex regulatory mechanisms are achieved through multivalency and avidity, neither of which are straightforward to realise with conventional peptide technologies. We show that tandemrepeat proteins possess all the necessary features with which to build such a platform
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