Engineering mono- and multi-valent inhibitors on a modular repeat-protein scaffold to target oncogenic Tankyrase

Tandem-repeat proteins are a class of proteins ubiquitous in nature and exploited in recent years in biotechnological and pharmaceutical applications due to their favourable biophysical properties. One such repeat motif, the tetratricopeptide repeat (TPR), has already been exploited for biotechnological applications and here the consensus-designed TPR (CTPR) sequence was used as a scaffold to create novel arrays of binding molecules. The platform allows us to display single and multiple functions with diverse geometrical arrangements by grafting short binding sequences onto the loops between adjacent repeats or at the terminal alpha-helices. As proof of concept, proteins were designed to bind to and inhibit the human tankyrase (TNKS), a key regulatory protein involved in Wnt signalling and overexpressed in cancer and other disorders. For this purpose, a tankyrase-binding peptide (TBP) was grafted between two adjacent repeats to create a solvent-exposed loop. A series of mono- and multi- valent TNKS binders, named TBP-CTPR, was assembled by repeating the TNKS-binding unit in tandem in both a monomeric format and also in a trimeric arrangement. The folding and thermodynamic stability of these TBP-CTPR proteins were characterised and the interaction with TNKS was measured using a range of biophysical approaches. Both the engineered TBP- CTPR proteins and TNKS are multivalent, and the effects of multivalency were explored both in the test tube and in the cell. The results show that the proteins interact to form large assemblies. Moreover, the TBP-CTPR proteins were found to have exceptional activity in inhibiting the Wnt signaling pathway upon delivery by encapsulation in fusogenic liposomes. Lastly, hetero-bifunctional constructs were generated by grafting two different binding sequences onto the CTPR scaffold, and a preliminary analysis of their activities was performed. In conclusion, these results point to the tremendous potential of the CTPR scaffold as a platform to build synthetic protein binders, with a particular focus on multivalent interactions.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2020

Author dc:creator

• Diamante, Aurora

Advisor dc:contributor.advisor

• Itzhaki, Laura

• Tankyrase
• repeat protein
• biotherapeutics

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/2d1f758f-2995-4a55-9984-3b9e879e8e70/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

Author Identifier

0000-0001-6504-2576

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/321246